Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.     

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.     

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.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

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.     

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.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The germ layer origin of mouse vaginal epithelium restricts its responsiveness to mesenchymal inductors: uterine induction

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the lower fused Müllerian ducts. While previously it has been reported that neonatal vaginal epithelium can be induced to differentiate as uterus, which normally develops from the middle portion of the Müllerian ducts, it has not been determined whether this ability is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test if germ layer origin influences the ability of vaginal epithelium to undergo uterine differentiation, we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with uterine mesenchyme, and grown them for 4 weeks in female mice. Mesoderm-derived Müllerian vaginal epithelium in combination with uterine mesenchyme formed the simple columnar epithelium typical of uterus. Similar results were obtained with neonatal cervical epithelium, another mesodermal Müllerian duct derivative. On the other hand, sinus vaginal epithelium combined with uterine mesenchyme formed small cysts lined by a stratified squamous vaginal-like epithelium. This epithelium never showed evidence of cycling between the cornified and mucified states as is typically seen in vaginal epithelium combined with vaginal stroma. These results indicate that the ability of epithelium to form uterus is limited to mesoderm-derived epithelia and suggest that endoderm-derived sinus vaginal epithelium cannot undergo the typical differentiative modifications in response to the hormonal fluctuations of the estrous cycle when associated with uterine stroma.     

The use of protamine to study [6,7-3H]-oestradiol-17β binding in rat uterus

1. The binding of [6,7-(3)H]oestradiol-17beta to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17beta-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-(3)H]oestradiol-17beta with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-(3)H]testosterone sulphate or [1,2-(3)H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-(3)H]oestradiol-17beta, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-(3)H]oestradiol-17beta. None of these procedures affected the dissociation constant K(d) or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17beta, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-(3)H]oestradiol-17beta to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.     

Essential roles of mesenchyme-derived beta-catenin in mouse Müllerian duct morphogenesis

Members of the Wnt family of genes such as Wnt4, Wnt5a, and Wnt7a have been implicated in the formation and morphogenesis of the Müllerian duct into various parts of the female reproductive tract. These WNT ligands elicit their action via either the canonical WNT/beta-catenin or the non-canonical WNT/calcium pathway and could possibly function redundantly in Müllerian duct differentiation. By using the Müllerian duct-specific anti-Müllerian hormone receptor 2 cre (Amhr2-cre) mouse line, we established a conditional knockout model that removed beta-catenin specifically in the mesenchyme of the Müllerian duct. At birth, loss of beta-catenin in the Müllerian duct mesenchyme disrupted the normal coiling of the oviduct in the knockout embryo, resembling the phenotype of the Wnt7a knockout. The overall development of the female reproductive tract was stunted at birth with a decrease in proliferation in the mesenchyme and epithelium. We also discovered that Wnt5a and Wnt7a expression remained normal, excluding the possibility that the phenotypes resulted from a loss of these WNT ligands. We examined the expression of Frizzled (Fzd), the receptors for WNT, and found that Fzd1 is one receptor present in the Müllerian duct mesenchyme and could be the putative receptor for beta-catenin activation in the Müllerian duct. In summary, our findings suggest that mesenchymal beta-catenin is a downstream effector of Wnt7a that mediates the patterning of the oviduct and proper differentiation of the uterus.     

Characterization of rat hypothalamic progestin binding by spheroidal hydroxylapatite chromatography.

The progestin-high-affinity-binding components in rat target tissues have been assayed by a simple and precise procedure by using spheroidal hydroxylapatite. The progestin 'receptors' in the uterus and hypothalamus of female rats are highly specific for progestins, which they bind with high affinity (Kd for [3H]progesterone in hypothalamus is 1.9 nM and in uterus is 3.7 nM). The dissociation of [3H]progesterone from receptor in vitro is rapid: t1/2 6 degrees C = 45 min in uterine cytosol; t1/2 6 degrees C = 160 min in hypothalamic cytosol. The binding is destroyed by proteinase. In the cytosol of hypothalamus and cortex of developing rats, progestin 'receptors' were present in both male and female rats by 2-3 days after birth; subsequent changes in concentration of these 'receptors' appeared to be independent of sex. Concentrations of progestin 'receptor' were close to adult values by 8-9 days, and thereafter changed relatively little.     

Characterization of R5020 and RU486 binding to progesterone receptor from calf uterus

We have examined and compared the binding characteristics of the progesterone agonist R5020 [promegestone, 17,21-dimethylpregna-4,9(10)-diene-3,20-dione] and the progesterone antagonist RU486 [mifepristone, 17 beta-hydroxy-11 beta-[4-(dimethylamino) phenyl]-17 alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] in calf uterine cytosol. Both steroids bound cytosol macromolecule(s) with high affinity, exhibiting Kd values of 5.6 and 3.6 nM for R5020 and RU486 binding, respectively. The binding of the steroids to the macromolecule(s) was rapid at 4 degrees C, showing saturation of binding sites at 1-2 h for [3H]progesterone and 2-4 h for both [3H]R5020 and [3H]RU486. Addition of molybdate and glycerol to cytosol increased the extent of [3H]R5020 binding. The extent of [3H]RU486 binding remained unchanged in the presence of molybdate, whereas glycerol had an inhibitory effect. Molybdate alone or in combination with glycerol stabilized the [3H]R5020- and [3H]RU486-receptor complexes at 37 degrees C. Although the rate of association of [3H]RU486 with the cytosolic macromolecule was slower than that of [3H]R5020, its dissociation from the ligand-macromolecule complex was significantly slower than [3H]R5020. Competitive steroid binding analysis revealed that [3H]progesterone, [3H]R5020, and [3H]RU486 compete for the same site(s) in the uterine cytosol, suggesting that all three bind to the progesterone receptor (PR). Sedimentation rate analysis showed that both steroids were bound to a molecule that sediments in the 8S region. The 8S [3H]R5020 and [3H]RU486 peaks were abolished by excess radioinert progesterone, RU486, or R5020.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

Studies on sex-organ development. Prenatal effect of oestrogenic hormone on tubular-gland cell morphogenesis and ovalbumin-gene expression in the chick Müllerian duct.

The effects of diethylstilboestrol on morphogenesis and cyto-differentiation of the chick-embryo left Müllerian duct were examined. Embryos were treated at different stages of development with maximal-responsive doses of diethylstilboestrol over a 5-day interval. The shell gland and magnum regions of the Müllerian duct were then assayed for growth and histological morphogenesis. The results were correlated with diethylstilboestrol-induced ovalbumin-gene expression as measured by ovalbumin-mRNA (mRNAov) accumulation and the relative rate of ovalbumin synthesis. Treatment of the embryo from day 10 to day 15 of incubation induces morphogenesis of tubular-gland cells in the Müllerian-duct magnum. Although these cells constitute 10% of the total cell population and contain an average of 8000 molecules of mRNAov per cell, ovalbumin synthesis is only 0.85% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 13 to day 18 of incubation contains about 30% tubular-gland cells, which have accumulated an average of 7000 molecules of mRNAov per cell, but ovalbumin synthesis is only 3.25% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 16 to day 21 of incubation contains about 50% tubular-gland cells, which have accumulated an average of 6500 mRNAov molecules per cell, and ovalbumin synthesis is 10% of total magnum protein synthesis. Oestrogen responsiveness develops simultaneously in the Müllerian-duct magnum and shell-gland regions. Compared with the rate of diethylstilboestrol-induced oviduct growth, the relative rate of diethylstilboestrol-induced Müllerian-duct growth increases with embryonic age, from 20-fold lower in the 10-day embryo to only 3-fold lower in the 16-day embryo. All results are discussed in comparison with the responses to oestrogen of the immature chick oviduct, and in terms of the ontogeny of hormone-competent epithelial and stromal components of the Müllerian duct. It is concluded that the development of oestrogenic competence in the embryonic Müllerian duct is a multiphasic phenomenon. A dramatic increase in hormone responsiveness in the Müllerian duct occurs between days 10 and 16 of development, and a less dramatic final maturation of oestrogen responsiveness occurs between day 16 of development and 1 week after hatching.     

Conditional deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a switch to adipogenesis in the myometrium

Precise cell fate decisions during differentiation of uterine tissues from the embryonic Müllerian duct are critical for normal fertility. Wnt-7a, a member of the Wnt family of secreted signaling molecules that can signal through a canonical beta-catenin pathway, is necessary for the correct differentiation of both anterior/posterior and radial axes of the uterus. In order to investigate the role of beta-catenin directly in mouse uterine development, we have generated mice that are deficient in beta-catenin expression in the embryonic Müllerian duct. We have found that conditional deletion of beta-catenin in the Müllerian duct mesenchyme before postnatal differentiation of the uterine layers results in a phenotype that is distinct from the phenotype observed by deletion of Wnt-7a. Shortly after birth, the uteri of the conditional mutants appear smaller and less organized. The uteri of adult conditional beta-catenin mutants are grossly deficient in smooth muscle of the myometrium, which has been replaced by adipose, a phenotype resembling human lipoleiomyoma. We also show that the adipocytes in the uteri of mice conditionally deleted for beta-catenin are derived from Müllerian inhibiting substance type II receptor-expressing cells suggesting that they share a common origin with the uterine smooth muscle cells. These results describe the first molecular evidence linking disruption of beta-catenin expression in mesenchymal cells with a switch from myogenesis to adipogenesis in vivo.     

Anti-Müllerian hormone in three intersex conditions.

Anti-Müllerian hormone (AMH), secreted by embryonic testicular Sertoli cells, inhibits the development of Müllerian ducts in the male. An enzyme-linked immunoassay (ELISA) for AMH was used to investigate three intersex infants. The AMH level was correlated with each patient's degree of Müllerian duct development. Complete inhibition of Müllerian structures correlated with the normal levels of AMH in the infant with testicular feminization. Detectable levels of AMH were found in the hermaphroditic infant; however, these low levels reflected Sertoli cell inadequacy of the ovotestis, which was documented by a right rudimentary Fallopian tube and a normal uterus. In the infant with persistent Müllerian duct syndrome, (PMDS), the normal Müllerian derivatives are compatible with 1) an AMH receptor defect; 2) a biologically and immunologically abnormal AMH molecule, or 3) a functional AMH deletion. The lack of detectable AMH in this infant excluded the AMH receptor abnormality and thus directed authors' search for the specific defect to the AMH gene. Thus, this ELISA for AMH is as valuable a tool to the molecular biologist studying a precise genetic error as it is to the physician making a precise clinical diagnosis.     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.