Landau renormalizations of superfluid density in the heavy-fermion superconductor CeCoIn5.

The formation of heavy-fermion bands can occur by means of the conversion of a periodic array of local moments into itinerant electrons via the Kondo effect and the huge consequent Fermi-liquid renormalizations. Leggett predicted for liquid (3)He that Fermi-liquid renormalizations change in the superconducting state, leading to a temperature dependence of the London penetration depth Λ quite different from that in BCS theory. Using Leggett's theory, as modified for heavy fermions, it is possible to extract from the measured temperature dependence of Λ in high quality samples both Landau parameters F(0)(s) and F(1)(s); this has never been accomplished before. A modification of the temperature dependence of the electronic specific heat C(el), related to that of Λ, is also expected. We have carefully determined the magnitude and temperature dependence of Λ in CeCoIn(5) by muon spin relaxation rate measurements to obtain F(0)(s) = 36 ± 1 and F(1)(s) = 1.2 ± 0.3, and we find a consistent change in the temperature dependence of C(el). This, the first determination of F(1)(s) with a value ≪ F(0)(s) in a heavy-fermion compound, tests the basic assumption of the theory of heavy fermions, that the frequency dependence of the self-energy is much more important than its momentum dependence.

Direct search for a ferromagnetic phase in a heavily overdoped nonsuperconducting copper oxide.

The doping of charge carriers into the CuO(2) planes of copper oxide Mott insulators causes a gradual destruction of antiferromagnetism and the emergence of high-temperature superconductivity. Optimal superconductivity is achieved at a doping concentration p beyond which further increases in doping cause a weakening and eventual disappearance of superconductivity. A potential explanation for this demise is that ferromagnetic fluctuations compete with superconductivity in the overdoped regime. In this case, a ferromagnetic phase at very low temperatures is predicted to exist beyond the doping concentration at which superconductivity disappears. Here we report on a direct examination of this scenario in overdoped La(2-x)Sr(x)CuO(4) using the technique of muon spin relaxation. We detect the onset of static magnetic moments of electronic origin at low temperature in the heavily overdoped nonsuperconducting region. However, the magnetism does not exist in a commensurate long-range ordered state. Instead it appears as a dilute concentration of static magnetic moments. This finding places severe restrictions on the form of ferromagnetism that may exist in the overdoped regime. Although an extrinsic impurity cannot be absolutely ruled out as the source of the magnetism that does occur, the results presented here lend support to electronic band calculations that predict the occurrence of weak localized ferromagnetism at high doping.

Direct observation of the quantum critical point in heavy fermion CeRhSi3.

We report on muon spin rotation studies of the noncentrosymmetric heavy fermion antiferromagnet CeRhSi3. A drastic and monotonic suppression of the internal fields, at the lowest measured temperature, was observed upon an increase of external pressure. Our data suggest that the ordered moments are gradually quenched with increasing pressure, in a manner different from the pressure dependence of the Néel temperature. At 23.6 kbar, the ordered magnetic moments are fully suppressed via a second-order phase transition, and T(N) is zero. Thus, we directly observed the quantum critical point at 23.6 kbar hidden inside the superconducting phase of CeRhSi3.

Expression of anti-Müllerian hormone (AMH) in equine granulosa-cell tumors and in normal equine ovaries.

Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is expressed by granulosa cells in females of many mammalian species, and circulating AMH concentrations have been used to monitor granulosa-cell tumors (GCT) in women. The objective was to characterize expression of AMH in equine GCT, and in normal equine ovaries, based upon immunohistochemistry (IHC), using a polyclonal primary antibody directed against human AMH. Equine GCT (n=27) and normal equine ovaries (n=10) were examined by IHC. In addition, sera from four mares with GCT were characterized for AMH bioactivity, based upon suppression of Müllerian duct development in the fetal rat. Immunolabeling with alpha-AMH was localized to granulosa cells in equine GCT, as well as within antral follicles in normal ovaries. Expression of AMH first appeared in granulosa cells of small growing follicles and was most intense in small antral follicles; large antral or atretic follicles had reduced immunolabeling. Omission of the primary antibody or incubation of the primary antibody with the corresponding blocking peptide eliminated immunolabeling of granulosa cells in GCT and in normal antral follicles, confirming the specificity of the immunolabel. Sera from mares with GCT had increased AMH bioactivity compared to control sera. In conclusion, AMH was strongly expressed by granulosa cells in equine GCT and in normal antral follicles. Therefore, anti-Müllerian hormone may be a useful biomarker for detection of GCT in the horse.

A time-less function for mouse timeless.

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo.

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.

Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.

Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.

Specific estradiol binding in embryonic Mullerian ducts: a potential modulator of regression in the male and female chick.

The total content of putative estradiol-specific cytosolic type I and nuclear type I and II estradiol-specific binders was measured in 8- and 9-day-old male and female chick embryo Mullerian ducts. Cytosolic and nuclear type I estradiol-specific binding levels were similar in males and females, and no significant differences were noted among right vs. left and 8-day-old vs. 9-day-old embryo Mullerian ducts. The levels of nuclear type I estradiol binder were consistently higher than the cytosolic type I binder, but this difference was not significant. Nuclear type II estradiol-specific binding, however, was significantly higher in the left Mullerian ducts of both male and female embryos. The significance of these findings in relation to the regression of Mullerian ducts in male and female chick embryos is discussed.

Mullerian inhibiting substance in the adult rat ovary during various stages of the estrous cycle.

Mullerian inhibiting substance (MIS) in the adult rat ovary can be detected by immunohistochemistry in the granulosa cells of growing preantral follicles and in the cumulus oophorus and periluminal granulosa cells of antral follicles in estrus, metestrus, diestrus, and proestrus. Neither the corpus luteum nor atreitic follicles stained for MIS. During proestrus, dramatic changes occurred in the large preovulatory antral follicles, which early in the day manifest intense MIS-specific staining in the granulosa cells located close to the oocyte, however, at 2300 h, just before ovulation when the germinal vesicle was extruded to indicate resumed meiotic division, MIS staining waned. When the morphology of the late preovulatory stage was created experimentally in 26-day-old immature ovaries by stimulating 48 h earlier with hCG, the intense staining of the granulosa cells surrounding the oocytes from untreated ovaries was lost in the cumulus cells of such hCG-treated animals. This temporal pattern of MIS staining and the prior demonstration that MIS could inhibit in vitro meiosis of oocytes from untreated immature rats suggest that this regressor, well characterized in the fetal testis, might function in the ovary as a regulator of oocyte maturation and follicular development during the adult reproductive cycle.

Detection of Müllerian inhibiting substance in biological samples by a solid phase sandwich radioimmunoassay.

Müllerian inhibiting substance (MIS) is a 140,000-dalton glycoprotein responsible for regression of Müllerian ducts in a male embryo. It has recently been demonstrated that MIS inhibits the growth of tumors in vivo and in vitro. In this study, we have constructed a sensitive, solid phase sandwich RIA using monoclonal antibodies raised to bovine MIS. The amount of MIS detected was based on the protein concentration of Green 3, the most purified fraction of MIS available. The assay could detect 20 ng Green 3 or 0.14 pmol in physiological samples. There was no evidence of cross-reactivity of the antibodies raised to bovine MIS with chicken, rat, mouse, or human MIS.

Identification of a receptor for human müllerian inhibiting substance.

Müllerian inhibiting substance (MIS), a Sertoli cell-derived glycoprotein and member of the transforming growth factor-beta supergene family, plays a key down-stream role in mammalian sex determination. Identification of a receptor for MIS has now been achieved in a MIS-responsive human vulvar carcinoma cell line, A431, using fluorescein isothiocyanate labeling of recombinant human MIS (FITC-rhMIS) and RRAs with iodinated carboxy-terminal rhMIS. Confocal fluorescence microscopy of A431 cells incubated on ice with 30-nM concentrations of covalently bound FITC-rhMIS reveals specific punctate cell surface fluorescent signal. Cytosolic fluorescent signal is seen after incubation at 37 C for 1 h as well as occasional apparent perinuclear accumulation. FITC-rhMIS coincubated with molar excesses of unlabeled rhMIS in A431 cells eliminates cell surface and cytosolic fluorescent uptake. Double label experiments with FITC-rhMIS and tetramethyl rhodamine isothiocyanate epidermal growth factor establish separate binding of each ligand, displaceable, respectively, by large molar excesses of unlabeled rhMIS or epidermal growth factor. RRAs reveal a single, high affinity (Kd, 5.8 nM), saturable, low abundance binding species for carboxy-terminal rhMIS. Solubilized supernatants of A431 whole cells cross-linked with 125I-carboxy-terminal rhMIS identify a band with a mol wt of 88,000 on electrophoresis and autoradiography. This identification of a MIS receptor in A431 cells now permits the design of affinity purification protocols using rhMIS, followed by direct protein microsequencing.

Mullerian duct regression and antiproliferative bioactivities of mullerian inhibiting substance reside in its carboxy-terminal domain.

A 25-kilodalton dimeric carboxy-terminal fragment of the recombinant human Mullerian inhibiting substance protein (rhMIS) was produced by proteolytic cleavage with plasmin and purified by size-exclusion chromatography. The identity of the isolated dimer as the carboxy-terminal fragment was confirmed by gel electrophoresis and Western analysis. As was true of every sample of the holo molecule, all preparations of the carboxy-terminal domain of rhMIS (n = 10), when added in the 0.5-5.0 micrograms/ml range, exhibited a dose-dependent partial to complete regression of the 14.5-day fetal rat Mullerian duct in an organ culture assay. The carboxy-terminal dimer also inhibited, in a dose-dependent manner, the growth of A431 cells in monolayer cultures. Daily addition of 5, 10, or 20 micrograms carboxy-terminus for 3 days resulted in 0%, 25%, and 100% inhibition of cell proliferation, respectively. Similar and higher doses of holo rhMIS had no or inconsistent antiproliferative activity (0-34% inhibition), even though the preparations caused Mullerian duct regression. All amino-terminal fragments prepared using this separation protocol were found to be inactive in these assays. These findings suggest that the bioactivity of rhMIS as a regressor of fetal Mullerian ducts and an inhibitor of A431 cell growth resides in its carboxy-terminal domain. These results indicate that the urogenital ridge tissue, but not A431 cells in culture, may be capable of cleaving intact MIS to a biologically active conformation.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Developmental expression of a candidate müllerian inhibiting substance type II receptor.

We have isolated a candidate Müllerian inhibiting substance (MIS) type II receptor complementary DNA from an embryonic rat urogenital ridge library and have studied its binding to MIS, its developmental pattern of expression and tissue distribution. By in situ hybridization with a full-length riboprobe, the receptor is expressed in the mesenchymal cells surrounding the Müllerian duct at embryonic days 14, 15, and 16 and in tubular and follicular structures of the rat fetal gonads. Expression of the messenger RNA was also seen in the granules cells and seminiferous tubules of pubertal gonads. Northern analysis revealed that the MIS type II receptor messenger RNA is highly expressed in embryonic, pubertal, and adult testes and ovaries, as well as in the gravid uterus. The timing of expression in the gonads of both sexes was also analyzed by Northern analyses that showed high levels of expression at the time of Müllerian duct regression, much lower levels neonatally and prepubertally and then increased expression again with sexual maturation. The tissue and developmental specificity of expression of this receptor, which make it likely that this is the functional MIS type II receptor, can be used to advantage in therapeutic targeting strategies and to decipher the function of MIS in the gonads.

Progesterone binding by normal and abnormal human endometrium.

Cytosols from 75 normal, 36 abnormal, and 5 decidual human endometrial tissue specimens were assayed for the presence of a high affinity, progesterone-specific binding protein. Thirty of the normal and 15 of the abnormal samples were found to contain a binder which would form a high-affinity complex with progesterone but not with cortisol, 17beta-estradiol, testosterone, or 5alpha-androstane-3-,17-dione. Incubation of cytosol with trypsin or incubation for 2 hours at 37 C abolished [3H]progesterone binding by these preparations, indicating the protein nature and heat-lability of the binder. The average equilibrium constant of dissociation, Kd, of the progesterone-binder complex was 4.0 X 10(-10)M in each phase of the menstrual cycle. The concentration of the binder varied over the cycle, however, with a significant peak at mid-cycle (P = .02). The average saturation values in femtomoles (fmoles)/mg protein ranged from 21 in the early proliferative phase to 64 in the late proliferative samples, dropping to 36 in early secretory and to 3 in the late secretory phase of the cycle. No progesterone-specific binding was detected in decidual samples. Saturable binding was demonstrable in 10 of 22 endometrial hyperplasias, 80-1840 fmoles/mg protein, with high affinity, Kd 3.3 X 10(-10)M. Two other hyperplasia samples bound progesterone, but with lower affinity. Two grade I adenocarcinomas, one grade III adenosquamous carcinoma, and one grade III adenocarcinoma contained the progesterone binder, but in 9 other cancers no detectable binder was present. A benigh adenocanthomyoma was found to contain a progesterone binder (18 fmoles/mg protein with a Kd of 2.5 X 10(-10)M).

Lack of evidence for estrogen receptors in human and bovine parathyroid tissue.

It has been hypothesized that estrogen may have a direct effect to reduce the set-point for PTH secretion which may be implicated in its hypocalcemic action. If this is so, estrogen receptors should be demonstrable within parathyroid tissue. Seven human parathyroid adenomas and five samples of normal bovine parathyroid tissue were examined using classical hormone-binding techniques. In no case was there evidence of displaceable estrogen binding of high affinity and low capacity. To exclude the presence of receptors within a small subset of the cells, five of the human adenomas were further studied by immunohistochemistry using a monoclonal antibody to the human estrogen receptor. In no case was there evidence of estrogen receptors. We conclude that the hypocalcemic action of estrogen replacement is unlikely to be mediated via a classical estrogen receptor within the parathyroid, although normal parathyroid tissue was not studied.

Gonadotropin-releasing hormone agonist-induced differences in granulosa cell cycle kinetics are associated with alterations in follicular fluid müllerian-inhibiting substance and androgen content.

We have previously shown that the proliferative index (PI), as determined by flow cytometry of luteinized granulosa cells obtained at oocyte retrieval, is greater in ovulation induction regimens which include the GnRH analog (GnRH-a) leuprolide acetate than those using human menopausal gonadotropin (hMG) only. Specific growth factors or intrafollicular hormones may contribute to this leuprolide acetate-induced difference in cell cycle kinetics. We examined whether differences in the PI of these granulosa cells are associated with the alterations of follicular fluid content of Mullerian-inhibiting substance (MIS) and other intrafollicular hormones including FSH, estradiol, progesterone, androstenedione, and testosterone. The control group consisted of follicular fluid obtained from 18 follicles from 4 women receiving hMG alone. The GnRH-a treated group consisted of follicular fluids obtained from 55 follicles aspirated from 18 women receiving GnRH-a in addition to hMG. One-way analysis of variance using log-transformed data and expressed as geometric means with 95% confidence intervals, demonstrated that the follicles from the control group had a significant 14-fold higher concentration of 2.46 ng/mL MIS, 95% CI (1.8-4.8) vs. 0.18 ng/mL, 95% CI (0.13-0.24) P < 0.0005, a 3-fold higher concentration of 17.55 nmol/L androstenedione, 95% CI (14.6-20.9) vs. 5.76 nmol/L, 95% CI (3.1-10.5) P < 0.02, and a 1.5-fold higher concentration of 29.43 nmol/L testosterone 95% CI (22.5-38.14) vs. 19.3 nmol/L, 95% of CI (11.1-33.9) P < 0.01 than GnRH-a treated follicles, although the PI value in controls was half that of the GnRH-a group. These data demonstrate that GnRH-a induced differences in granulosa cell cycle kinetics are associated with alterations of MIS and androgen intrafollicular fluid content and suggest that MIS may be a mitotic inhibitor of human granulosa cells.

The inhibitory effects of müllerian-inhibiting substance on epidermal growth factor induced proliferation and progesterone production of human granulosa-luteal cells.

This study was undertaken to determine if Müllerian-inhibiting substance (MIS) could block basal and/or epidermal growth factor (EGF)-induced proliferation and progesterone production by cultured human granulosa-luteal cells. Cells from follicles of individual patients were pooled, counted, and aliquoted into Ham's F-10 medium containing 10% MIS-free female fetal calf serum at 37 C in 95% air and 5% CO2. After assessing viability, cells were counted on days 4, 8, 12, and 16 of culture. EGF was added every other day at 0.2, 2, and 20 ng/mL beginning on culture day 4. The greatest stimulatory effect of EGF on cell proliferation was observed at 20 ng/mL on days 12 and 16. EGF increased progesterone production per cell after 4 days exposure, but this effect was lost after 8 days. Granulosa-luteal cells were cultured with 0.2, 2, and 20 ng/mL immunoaffinity purified recombinant human MIS (rhMIS) or conditioned medium from Chinese hamster ovary cells transfected with the human MIS gene, beginning on culture day 4. rhMIS demonstrated its greatest inhibitory effect on cell proliferation at 20 ng/mL on day 16. The rhMIS decreased progesterone production per cell after 4 days exposure, but only in the higher doses. Maintaining EGF at 20 ng/mL and varying rhMIS yielded significant reduction in EGF-mediated proliferation and progesterone production per cell at 2 and 20 ng/mL rhMIS. These experiments demonstrate rhMIS inhibits basal and EGF-stimulated human granulosa-luteal cell proliferation and progesterone production.

Mullerian inhibiting substance in humans: normal levels from infancy to adulthood.

Mullerian-inhibiting substance (MIS) is a gonadal hormone synthesized by Sertoli cells of the testis and granulosa cells of the ovary. To facilitate the use of MIS for the evaluation of intersex disorders and as a tumor marker in women with MIS-expressing ovarian tumors, we measured MIS in 600 serum samples from males and females. These data show that mean MIS values for males rise rapidly during the first year of life and are highest during late infancy, then gradually decline until puberty. In contrast, MIS values in females are lowest at birth and exhibit a minimal increase throughout the prepubertal years. Whereas MIS is uniformly measurable in all prepubertal boys studied, it is undetectable in most prepubertal female subjects. These data reveal an easily discernible sexually dimorphic pattern of expression and confirm that MIS can be used as a testis-specific marker during infancy and early childhood. MIS values that are above the upper limits for females are discriminatory for the presence of testicular tissue or ovarian tumor, and those below the lower limits for males are consistent with dysgenetic or absent testes or the presence of ovarian tissue. These data will enable normal and abnormal levels of MIS to be differentiated with higher precision and will facilitate the use of MIS in the management of gonadal disorders.