Sexually dimorphic expression of a teleost homologue of Müllerian inhibiting substance during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein belonging to transforming growth factor beta superfamily. In mammals, MIS is responsible for regression of Müllerian ducts, anlagen of the female reproductive ducts, in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fishes, which do not have the Müllerian ducts, has yet to be clarified. To address the role of MIS on gonadal sex differentiation in fishes, we isolated a MIS cDNA from the Japanese flounder testis and examined the expression pattern of MIS mRNA in gonads of both sexes during sex differentiation period. In this study, we present the first demonstration of sexually dimorphic expression of MIS mRNA during sex differentiation in teleost fishes, similarly to amniote vertebrates which possess the Müllerian ducts.     

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.     

Cellular mechanisms of Müllerian duct formation in the mouse

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

Androgens and the development of the vagina

Today it is generally held that the vagina develops from sinovaginal bulbs and that the lower third of the definitive vagina is derived from the urogenital sinus. Here we show that the entire vagina arises by downward growth of Wolffian and Müllerian ducts, that the sinovaginal bulbs are in fact the caudal ends of the Wolffian ducts, and that vaginal development is under negative control of androgens. We designed a genetic experiment in which the androgen receptor defect in the Tfm mouse was used to examine the effects of androgens. Vaginal development was studied by 3D reconstruction in androgen-treated female embryos and in complete androgen-insensitive littermates. In androgen-treated females, descent of the genital ducts was inhibited, and a vagina formed in androgen-insensitive Tfm embryos as it does in normal females. By immmunohistochemical localization of the androgen receptor in normal mouse embryos, we demonstrated that the androgen receptor was expressed in Wolffian duct and urogenital sinus-derived structures, and was entirely absent in the Müllerian duct derivatives. We conclude that the Wolffian ducts are instrumental in conveying the negative control by androgens on vaginal development. The results are discussed under evolutionary aspects at the transition from marsupial to eutherian mammals.     

Immunohistochemical localisation of androgen receptor during sex-specific morphogenesis in the fetal mouse

The distribution of androgen receptors (ARs) in paraffin serial sections of day 17 and day 18 male and female mouse embryos was investigated. In the cranial section of the genital tract AR expression was restricted to Wolffian structures while Müllerian ducts and surrounding mesenchyme were AR negative. In the fusion zone with the urogenital sinus the epithelial components of the vaginal bud were clearly distinguished by differential AR expression, which was faint in the Wolffian ducts, totally missing in the Müllerian ducts, and intense in the sinus ridges with the most intense expression in the morphogenetically active mesenchyme, indicating a new mechanism of negative control of vagina formation via androgens. Expression of ARs outside the genital tract was observed: (1) in loose interstitial mesenchyme extending into the retroperitoneal space up to the coeliac artery, indicating androgen effects during ascent of the kidneys and descent of intraperitoneal organs, (2) in the trigone of the bladder indicating androgen involvement in the development of the vesico-ureteral junction, and (3) in loose mesenchyme between striated muscle fibres and around pelvic skeletal elements, indicating mediation of androgen effects on the musculoskeletal system via loose mesenchyme.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Müllerian inhibiting substance in reproduction and cancer.

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

Differentiation of the müllerian (paramesonephric) duct epithelium in the rat

Differentiation of the Müllerian duct epithelium was studied in 15- to 21-day female rat foetuses. The proximal segment of the Müllerian duct is formed by the 15th day; it runs parallel to the Wolffian duct and the two are wrapped in a common basement membrane. On the 16th day the genital ducts are clearly separate; the Müllerian duct has a slit-like lumen and is lined with simple columnar epithelium. Throughout the whole of the given period the epithelium retains a relatively indifferent appearance. Characteristic findings from the 18th day include the apical migration of centrioles and the formation of solitary cilia.     

St?rungen der m?nnlichen Gonadendifferenzierung

XY gonadal dysgenesis is characterized by a failure of testis differentiation and can be caused either by disturbed development of the urogenital ridge to the bipotential gonad or by impaired differentiation of the bipotential gonad to testis. Genes responsible for early gonadal development like WT1 and SF1 can be distinguished from genes involved in testis differentiation such as SRY, SOX9, DMRT, DAX1, WNT4, DHH, CBX2, TSPYL1, ATRX and ARX. In complete XY gonadal dysgenesis, M??llerian but no Wolffian structures are present. In partial XY gonadal dysgenesis, remnants of M??llerian and Wolffian ducts can be present and virilization of the external genitalia can take place. In about a third of cases, XY gonadal dysgenesis occurs in a syndromic form. In these syndromic forms, the extragenital phenotypes can indicate the causative genes, but these genes can also cause non-syndromic forms of XY gonadal dysgenesis.     

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

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

Reduction of germ cell number induced in vitro by the testis in fetal rat ovary may be caused by factors other than anti-Müllerian hormone

Fetal testes explanted at 16.5 days and cultured with female genital tracts from 13.5-day-old rat fetuses strongly inhibited the Müllerian ducts and reduced the number of ovarian germ cells. Such a reduction was not obtained during cultures with testes from 13.5 days, even though they clearly inhibited Müllerian ducts. When testes from 16.5 days were cultured at distance from the female tracts only the loss of germ cells was observed. These results suggest that testes from 16.5 days produce a diffusible factor distinct from AMH and which reduces the number of germ cells in cultured ovaries.     

Experimental manipulation of sexual differentiation in wallaby pouch young treated with exogenous steroids

We have investigated the effects of androgen or oestrogen treatment of female or male tammar wallabies from the day of birth, when the gonads are histologically undifferentiated, to day 25 of pouch life, when the gonads and the Wolffian and Müllerian ducts have differentiated and the testes have migrated through the inguinal canal. Female tammars treated with testosterone propionate (24-50 mg kg-1 day-1) orally for 25 days had enlarged Wolffian and Müllerian ducts. Mammary and pouch development, however, was indistinguishable from that of control females. The treatment had no apparent effect on ovarian development, or on ovarian position in the abdomen. The phallus of males and females was similar in size, and neither experimental treatment had a significant effect on its size at day 25. Male tammars treated with oestradiol benzoate (1.2-2.5 mg kg-1 day-1) orally for 25 days had gross hypertrophy of the urogenital sinus. Testicular morphology was abnormal; many of the germ cells appeared necrotic, the seminiferous tubules were of reduced diameter, and there were few Leydig cells and increased amounts of fibrous tissue between the tubules. The cortex of these gonads contained some areas which had an ovarian appearance, lacking tubules and containing numerous germ cells. The Müllerian ducts of control males had regressed, but this was prevented by oestrogen treatment, suggesting an inhibition of either Müllerian Inhibiting Substance (MIS) production or its action. Normal testicular migration was inhibited in treated males; the testes remained high in the abdomen, similar in position to the ovaries of control females, whilst control males all had testes in the inguinal region. The gubernaculum and processus vaginalis of control males extended into the scrotum, but in treated males they terminated outside it. Oestrogen treatment had no effect on the size of the scrotum and did not induce mammary or pouch development. These experiments show that marsupials, like eutherians, have a dual hormonal control of Wolffian and Müllerian development. By contrast, the initial development of the mammary glands, pouch, gubernaculum and scrotum does not appear to be under hormonal control and is therefore likely to be autonomous and dependent on genotype.     

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.     

Human müllerian inhibitory factor messenger ribonucleic acid is hormonally regulated in the fetal testis and in adult granulosa cells

The regression of the Müllerian ducts (the embryologic precursor of uterus, vagina, and Fallopian tubes) in the male fetus is caused by Müllerian inhibitory factor (MIF), a glycoprotein produced by fetal Sertoli cells. Although this Müllerian duct involution is complete before midgestation, the amount of MIF mRNA did not vary among 25 human fetal testis samples from 13 to 25.8 weeks of gestation. In cultured 20-week human testis cells, cAMP increased MIF mRNA 8.3-fold, but the human gonadotropins FSH and CG had no effect. In cultured adult human granulosa cells, CG and cAMP increased MIF mRNA accumulation to 430% and 890%, respectively, but FSH had no effect. The expression and hormonal regulation of MIF mRNA in midgestation testes and in adult granulosa cells indicate that MIF has physiological roles in the human gonad other than Müllerian duct regression.     

Embryonic gonadal and sexual organ development in a small viviparous skink, Niveoscincus ocellatus

The majority of research into the timing of gonad differentiation (and sex determination) in reptiles has focused on oviparous species. This is largely because: (1) most reptiles are oviparous; (2) it is easier to manipulate embryonic developmental conditions (e.g., temperature) of eggs than oviductal embryos and (3) modes of sex determination in oviparous taxa were thought to be more diverse since viviparity and environmental sex determination (ESD)/temperature-dependent sex determination (TSD) were considered incompatible. However, recent evidence suggests the two may well be compatible biological attributes, opening potential new lines of enquiry into the evolution and maintenance of sex determination. Unfortunately, the baseline information on embryonic development in viviparous species is lacking and information on gonad differentiation and sexual organ development is almost non-existent. Here we present an embryonic morphological development table (10 stages), the sequence of gonad differentiation and sexual organ development for the viviparous spotted snow skink (Niveoscincus ocellatus). Gonad differentiation in this species is similar to other reptilian species. Initially, the gonads are indifferent and both male and female accessory ducts are present. During stage 2, in the middle third of development, differentiation begins as the inner medulla regresses and the cortex thickens signaling ovary development, while the opposite occurs in testis formation. At this point, the Müllerian (female reproductive) duct regresses in males until it is lost (stage 6), while females retain both ducts until after birth. In the later stages of testis development, interstitial tissue forms in the medulla corresponding to maximum development of the hemipenes in males and the corresponding regression in the females.     

Quantitative change in fibronectin in cultured müllerian mesenchymal cells in response to diethylstilbestrol and müllerian-inhibiting substance

I report on the synthesis of fibronectin in the developing chick Müllerian duct mesenchymal cells. Before the differentiation of female chick Müllerian duct, the amount of fibronectin in the cells of the right duct is 44% lower than in the left duct. While after differentiation, the amount of fibronectin in the right duct is 29% lower, as compared to the left duct. Estrogenic hormone diethylstilbestrol (DES) treatment was carried out at the 5th day of incubation when both female Müllerian ducts were undifferentiated. Three days after DES treatment, the regression of the right duct was prevented, and the amount of fibronectin was induced by 89%, while induction in the left duct was 11%. Eight days after DES administration, the amount of fibronectin in the right and left Müllerian duct was induced by 150 and 76%, respectively. After DES treatment in the male embryo, both Müllerian ducts were retained, and the capacity for fibronectin synthesis was preserved. Application of the indirect immunocytochemical labeling technique revealed Müllerian-inhibiting substance (MIS) binding sites on the membrane of the Müllerian mesenchymal cells. The addition of chick MIS in the culture medium reduced the amount of detectable fibronectin in the cultured mesenchymal cells. The synthesis of fibronectin in intestinal mesenchymal cells was not affected by DES or MIS.     

Paracrine-mediated apoptosis in reproductive tract development

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions.     

Wnt4 action in gonadal development and sex determination

Wnt4 is a growth factor involved in multiple developmental processes such as the formation of the kidney, adrenal, mammary gland, pituitary and the female reproductive system. During mammalian embryogenesis, Wnt4 is expressed in the gonads of both sexes before sex determination events take place and is subsequently down-regulated in the male gonad. Inactivation of the Wnt4 gene in mice has revealed that it is involved at several steps of female reproductive development. Wnt4 is implicated in Müllerian duct regression, the formation of sex-specific vasculature, the inhibition of steroidogenesis and in sex-specific cell migration events. A mouse model of sex-reversal has partially unravelled the molecular pathways in which Wnt4 operates during the development of the female reproductive system. However, the specific molecular mechanism of action of Wnt4 during gonadal development remains unknown. This and downstream signaling pathways involved in Wnt4 action during female gonad development are reviewed and models of Wnt4 action are proposed for Müllerian duct formation, sex-specific vasculature development, and sex determination events. Further identification of critical downstream effectors of the Wnt4 signaling pathway in mouse models and in patients with sex-reversal conditions could help in understanding sex-reversal pathologies in humans.     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.