Study on sex-organ development. Oestrogen-receptor translocation in the developing chick Müllerian duct.

After oestradiol administration in vivo, 87-95% of the initial concentration of oestradiol receptor in the cytoplasm of the embryonic-chick Müllerian-duct cell was translocated into the nucleus. The process of translocation depends on the amount of oestardiol administered in vivo. At 6 h after oestradiol administration in vivo, about 30% replenishment of the initial content of the cytosol receptor was observed in the cytoplasm. The Müllerian-duct nuclei, after exposure to non-radioactive oestradiol, exhibit saturable exchange with [3H]oestradiol in vitro. The exchange of oestradiol is temperature- and time-dependent. The optimal temperature and time for exchange are 37-41 degrees C and 2h respectively. The [3H]oestradiol-receptor complex extracted from the exchanged nuclei is present in 5-6S form, and its isoelectric point is 6.8. The number of nuclear oestradiol-binding sites of the developing Müllerian duct are 1.66, 2.22, 2.63, and 2.50 pmol/mg of DNA respectively for embryos of 10, 12, 15 and 18 days. The dissociation constants of the nuclear oestradiol receptor of the four observed developmental stages range from 3.0 to 3.1 nM.     

Decrease in the secretion of anti-Müllerian hormone by the chick testis during development

At the end of embryonic life the chick embryonic testis possesses a low anti-Müllerian activity, as evidenced by the grafting method to female hosts. The percentage of grafted embryos presenting a Müllerian duct regression is not increased by administration of an anti-estrogenic drug (tamoxifen). This observation does not favour the hypothesis according to which the low percentage of regression could be due to a protection of Müllerian ducts by estrogens from the host ovary. It shows rather that the anti-Müllerian hormone secretion actually decreases during development.     

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.     

Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage- and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression.     

Deficiencies of the müllerian ducts induced by norethindrone in the female chick embryo

Norethindrone produces two effects on müllerian ducts (MD) of female chick embryos. It induces the loss of the lower end of both ducts, as a result of a stop in their development, before 8 days. After 12 days NET causes regressions of the upper part of the MD particularly of the oviduct. NET like estrogens are the only known substances which present these both properties.     

Quantification of müllerian inhibiting substance in developing chick gonads by a competitive enzyme-linked immunosorbent assay

An immunoblotting method was used to purify a Müllerian-inhibiting substance (MIS)-specific antiserum. The serum was used to quantify the content of MIS in developing chick gonads by competitive enzyme-linked immunosorbent assay. From embryonic stages to the eleventh week after hatching, male chicken testes have a high content of MIS in the following two stages: (1) from the sixth to the eighth day and from the fourteenth to the twentieth day of incubation, and (2) from the second to the eighth week after hatching. The high content of MIS in the early embryonic stage is closely correlated with the natural pattern of Müllerian duct regression observed in the male embryo. From the sixth to the twelfth day of incubation, the female right ovary contains a higher content of MIS than that of the left ovary. Up to the fourteenth day of incubation, the content of MIS in the left ovary reaches maximum levels and then declines. The combination of MIS from right and left ovaries was found to be highest in the ninth to the fourteenth day of incubation, when the regression of the right Müllerian duct reached its highest peak. However, the question of the inability of MIS to cause regression of the female left Müllerian duct and the caudal part of the right duct is raised and discussed. The hypothesis that prenatal estrogenic hormone (diethylstilbestrol) protects the Müllerian duct has been reevaluated. It was found that estrogen does not reduce the MIS content in prenatally treated gonads.     

A mesenchymal perspective of Müllerian duct differentiation and regression in Amhr2-lacZ mice

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding beta-galactosidase (beta-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express beta-gal activity in an Amhr2-specific pattern. In the gonads, beta-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. beta-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, beta-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males beta-gal expression encircled the duct. beta-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal beta-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.     

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.     

Müllerian inhibiting substance is present in testes of dogs with persistent müllerian duct syndrome

Breeding studies in a strain of miniature schnauzer dogs with Persistent Müllerian Duct Syndrome (PMDS) indicate this syndrome is inherited as an autosomal recessive trait, as it is in man. Testes of neonatal dogs affected with PMDS and normal male littermates were examined for Müllerian Inhibiting Substance (MIS) production by immunohistochemistry and bioassay. MIS immunoactivity was detected in Sertoli cells of normal and affected pups using an avidin-biotin complex-enhanced method. Rat embryonic Müllerian ducts regressed when cocultured with testis fragments of both normal and affected pups in a graded organ culture bioassay, demonstrating that the MIS produced was bioactive. These findings indicate that Müllerian duct persistence in affected dogs is not due to a mutation in the structural gene for MIS, but rather, by inference, to a failure of response to MIS at the receptor level.     

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.     

Timing and irreversibility of Müllerian duct inhibition in the embryonic reproductive tract of the human male

A study was undertaken to determine (1) the effects of endogenous Müllerian inhibiting substance (MIS) on the developing human fetal genital tract; (2) the time in fetal life when MIS is first capable of inhibiting the growth of the embryonic Müllerian ducts; and (3) the reversibility of the effects of MIS on the developing male Müllerian ducts. Human fetal reproductive tracts were transplanted and grown for sustained periods in vivo in athymic nude mice. The genital tracts from 12 male human fetuses, ages 51 to 68 days postovulation, were grafted without their associated gonads into castrated murine hosts and grown for 30 to 70 days. Controls consisted of genital tracts from 8 female human fetuses, ages day 53 to 70 that were grown under identical conditions. Male specimens grew to approximately one-half the size of female specimens and disclosed varying degrees of inhibition of the Müllerian duct system from absence of the Müllerian ducts in older specimens (after Day 63) to poorly segregated segments of stroma as the mildest defect (less than Day 61). It is concluded that (1) MIS secretion by the embryonic testes probably begins before Day 51 of gestation; (2) the effects of MIS are progressive during the so-called critical window; (3) the effects of MIS are permanent; and (4) the mesenchyme is an important target of MIS.     

Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.     

Studies on sex-organ development. Ontogeny of cytoplasmic oestrogen receptor in chick Müllerian duct.

Oestradiol receptors were observed in the cytoplasm of the chick Müllerian duct at several embryonic stages. The sedimentation coefficients and the dissociation constants of the receptor protein remained unchanged throughout the various stages of development. Specific binding of cytoplasmic receptor to [3H]oestradiol assayed in vitro was shown to be saturable at concentration of 10nM or higher. The number of oeastradiol-binding sites on a per-cell basis increased linearly from day 8 to day 12 of incubation and then levelled off from day 12 to the fourth day after hatching. These results indicate that in the developing embryonic sex organ, the same receptor protein is present throughout prenatal development. The concentration of the oestradiol receptor increases and reaches a constant value, but the capacity for the receptor to interact with the hormone does not change.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

Studies on sex-organ development. Isolation and characterization of an oestrogen receptor from chick Müllerian duct.

An oestradiol-binding macromolecule was observed in the left Müllerian duct of the 15-day female chick embryo. The embryonic receptor binds oestradiol with a high affinity and low capacity, having a Kd of 3.2 X 10(-9)M and a maximal number of sites of 5.45 fmol/10(6) cells in the left Müllerian duct. The receptor is protein in nature, as suggested by its susceptibility to proteolysis; in addition, it is organ- and steroid-specific. Judging by glycerol-gradient analysis, the hormone receptors in the cytosol are present in 8S and 4.5S forms, and the 8S form could be dissociated into a 4.5S form in the presence of 0.5M-KCl. A 4.5-6S receptor could be extracted from the nuclei. Under physiological salt conditions, the embryonic receptors bind to DNA-cellulose and can be eluted when the salt concentration is increased to 0.5M-KCl. Determination by isoelectric focusing indicates that the isoelectric point is 5.8 for the 8S and 6.9 for the 4.5S receptor.     

Gene expression change in the Müllerian duct of the mouse fetus exposed to diethylstilbestrol in utero

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 microg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.