Marker proteins in the particulate fraction of third-trimester amniotic fluid.

The present clinical evaluation of fetal lung maturity relies largely on the determination of the amniotic surfactant phospholipids phosphotidylglycerol, lecithin, and sphingomyelin, but there are many false negatives as well as false positives among diabetics. The use of other components of lung surfactant, namely, the hydrophobic surfactant proteins (SPs) has long been suggested as an alternative to the classical assay, but tests based on the detection of immunoreactive SP-A have not proved superior or supplanted phospholipid ratios as an index. This report investigates the proteins in a fraction of third-trimester human amniotic fluid (the particulate fraction) enriched in the SP complexes that form the surfactant monolayer. The proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining and immunoblotting. Eight proteins are of particular interest. Three novel proteins (termed AFPP-1, AFPP-4, and AFPP-8) and the alpha-fetoprotein/human serum albumin complex (AFPP-7) can be detected throughout the 28- to 38-week gestational window. The protein that is referred to as AFPP-2 could be identified as SP-A on the basis of immunologic cross-reactivity as well as size and charge characteristics. The time course of appearance of AFPP-2 was also followed in patients with Rh isoimmunization syndrome and was found to be the same as that seen for SP-A. The SP-A was detected as at least five major charged isoforms with multiple subisoforms of different molecular weight and can be distinguished from a related set of proteins (AFPP-5) that appear with a different time course but are possible precursors. Two other proteins (AFPP-3, AFPP-6), which are detectable inconsistently bear some similarity to others reported previously but not extensively characterized. These results define both constant and variable proteins of the particulate fraction of the amniotic fluid and indicate that certain protein isoforms are changing throughout the third trimester. These data enhance the possibility of the utilization of these proteins as markers of lung maturity in conditions such as maternal diabetes.

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Temporal relation of ovulation to salivary and vaginal electrical resistance patterns: implications for natural family planning.

An independent assessment of the CUE Monitor (Zetek, Aurora, Colorado) as an ovulation predictor was made with emphasis on its potential role in "natural family planning". The device provides a digital measurement of the electrical resistance of saliva and vaginal secretions. Twenty-nine menstrual cycles from 11 regularly cycling women were monitored with basal temperatures, urinary LH, pelvic ultrasound and the CUE monitor. Patterns of peak salivary electrical resistance were able to predict ovulation on average 5.3 (+/- 1.9 SD) days in advance. Despite variations in total length of the follicular phase from cycle to cycle, the within-subject variation of this predictive interval was quite small. Nadirs in the electrical resistance of vaginal secretions occurred within 2 days of ovulation in all but one patient. Variation in this interval from cycle-to-cycle was small as well. We propose an algorithm for the use of these intervals in "natural family planning" that could safely reduce the monthly abstinence period of present methods. The simplicity, objectivity and consistency of this device could result in their greater general acceptance.

PIP: The use of the CUE monitor of resistance of saliva and vaginal secretions for predicting ovulation was assessed in 29 cycles from 11 women. The CUE probe is battery-operated, and gives a reading in units proportional to ohms, in 10 seconds. It is used twice daily, at the same time, at least 10 hours after coitus. Salivary resistance is a reflection of ACTH and aldosterone secretion, and rises a mean of 7.2 (range 5-8) days before ovulation. The oral "signal" is defined as the 2nd day after the peak in resistance. The vaginal "signal" is defined as the 4th day after the nadir in resistance. Actual days of the LH surge in experimental subjects were determined, in 3 consecutive cycles in 7 women, by urinary LH by kit, by ultrasound and by basal body temperature. Within-subject variations in length of follicular phase were quite small. An algorithm for use of the CUE device for "natural family planning", using abstinence for 9 days, not including menses and alternate days avoiding coitus to measure vaginal mucus more accurately, is suggested.

Plasma gonadotropin-releasing hormone profiles after intravenous and subcutaneous bolus injection in thin and obese women.

Other studies of plasma gonadotropin-releasing hormone profiles after bolus injection have revealed earlier, sharper peaks and higher blood gonadotropin-releasing hormone levels with the intravenous (IV) than with the subcutaneous route of administration. We used both routes to administer gonadotropin-releasing hormone by bolus injection to thin and obese subjects. Plasma gonadotropin-releasing hormone profiles after IV administration were similar in both groups. The subcutaneous route produced flatter, delayed, and lower peaks, an effect markedly exaggerated in obese subjects, who demonstrated 95% lower peak gonadotropin-releasing hormone levels compared with the IV route and 64% lower levels than thin subjects after subcutaneous administration. These findings may be relevant to therapeutic failures observed in obese subjects using the subcutaneous route.

Proteins associated with untransformed estrogen receptor in vitro. Perturbation of hydrophobic interactions induces alterations in quaternary structure and exposure of the DNA-binding site.

Estrogen receptors from calf uteri have been analyzed by high-performance size-exclusion chromatography, chromatofocusing, and DNA affinity chromatography using conditions designed to evaluate the relative contribution of hydrophobic interactions between the steroid-binding subunit and other receptor-associated proteins. The single large (untransformed) species of soluble estrogen-receptor consistently (n = 9) found in calf uteri displayed a rapid change in Stokes radius from 8.0 to 3.5 nm upon exposure to elevated ionic strengths (0.4 M KCl). However, equilibration of the estrogen-receptor complex into urea (up to 6 M) did not dissociate the untransformed receptor into the 3.5-nm receptor form (subunit) observed in hypertonic (0.4 M KCl) buffers. Exposure to 6 M urea did result in conversion of the untransformed receptor (8.0 nm) to a 6.0-6.5-nm receptor form not previously observed in either hypotonic or hypertonic buffers. In the presence of both 6 M urea and 0.4 M KCl, the untransformed estrogen-receptor complex was converted to a smaller receptor form intermediate in apparent size (4.5-5.0 nm) to that observed in 6 M urea or 0.4 M KCl alone. The formation of this 4.5-5.0-nm receptor form was partially estrogen dependent as determined by parallel analyses of unliganded receptor in urea/KCl buffer. The urea-induced change in apparent size (8 nm to 6.0-6.5 nm) at low ionic strength was accompanied by little or no detectable change in net surface charge as determined by chromatofocusing but a complete exposure of the DNA-binding site as evidenced by nearly quantitative interaction with DNA-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for bovine preproinsulin.

A cDNA library was prepared from poly(A+) RNA isolated from fetal bovine pancreas. Bacterial colonies were screened for sequences homologous to a rat preproinsulin I cDNA probe. Ten positive clones were selected at random and further studied. Northern blot analyses revealed that seven of these clones hybridized to a single RNA species, of approximately 400 nucleotides. Sequence analysis of one of these clones (pbI2885) revealed the entire structural region of bovine preproinsulin mRNA including a 72 nucleotide region encoding a signal peptide enriched in hydrophobic residues. The overall nucleotide homology between bovine and human preproinsulin mRNA was 76% for the preregion, 89% for the A chain, 83% for the B chain, and 68% for the C peptide (including a 15 nucleotide deletion).

Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment.

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea (+/- 0.4 M KCl) buffers and control buffers (+/- 0.4 M KCl) by chromatography through small columns of Sephadex G-25 or by dialysis at 0-6 degrees C. Equilibrium dissociation constants (Kd) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17 beta-[3H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol (Kd = 0.36 +/- 0.09 nM, n = 14) which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity (Kd = 3.45 +/- 0.86 nM, n = 6) to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. However, KCl did help prevent the reduction in binding capacity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification.(ABSTRACT TRUNCATED AT 250 WORDS)

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites.

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.

Evaluation of the OvuSTICK urinary luteinizing hormone kit in normal and stimulated menstrual cycles.

The rapid analysis of luteinizing hormone (LH) in urine would provide a useful clinical tool in the diagnosis and treatment of infertility in women. Urinary LH levels were measured in midday and evening specimens collected during 75 normal and stimulated menstrual cycles (55 women) using a rapid, visual, semiquantitative enzyme immunoassay dipstick test (OvuSTICK) and compared with basal body temperature (BBT) records, visualization of follicular collapse by daily ultrasonography, and serum hormone levels. In all 75 cycles studied, an LH surge (or its absence) in urine was associated with a biphasic (or monophasic) BBT record and/or serum progesterone. In addition, when serum and urine samples were obtained simultaneously, the day of the LH surge (or its absence) in the urine and serum correlated 100%. Discrepancies between ovulation as diagnosed by ultrasound and the LH surge in urine and/or serum in several patients suggested that individual factor(s) may affect the interpretation of ultrasound imaging. It appears that a simple, rapid, clinically reliable colorimetric method such as the OvuSTICK urinary LH test is an important parameter for predicting the time of ovulation.

Effect of purified lithospermic acid and its oxidation product on luteinizing hormone release in vitro.

Crude aqueous extracts of the plant Lithospermum ruderale have been shown to have antigonadotropic activity that resides in its polyphenolic fractions. This study examined the ability of one such polyphenol, lithospermic acid (LA), and its oxidation product(s) (oxyLA) to inhibit luteinizing hormone (LH) secretion in vitro. Primary pituitary cultures were exposed for 4.5 or 6 h to either LA or oxyLA. In the presence of gonadotropin-releasing hormone (GnRH), oxyLA was at least 10 times more potent than LH in inhibiting LH release. In the absence of GnRH, oxyLA but not LA caused an increase in LH release. After washing to remove the oxyLA and LA, cultures were challenged with GnRH. Only cultures pretreated with oxyLA showed a decrease in GnRH-stimulated LH release. These results indicate that oxyLA may contain the primary antigonadotropic agents in L. ruderale. The different responses observed in the presence and absence of GnRH, and the morphologic features of the oxyLA-treated cultures, suggest that the mechanism of action may involve the cell membrane of the gonadotrope.

Isolation of unliganded steroid receptor proteins by high-performance size-exclusion chromatography. An investigation of steroid-dependent structural alterations.

The physicochemical properties of unliganded steroid receptor proteins remain largely unknown primarily due to receptor lability in the absence of specific ligand, especially during prolonged biochemical analyses. We have utilized high-performance size-exclusion chromatography (HPSEC) as a rapid means of investigating the structural properties of cytosolic estrogen receptor proteins in both the presence and absence of [3H]estradiol-17 beta. Cytosols prepared from immature calf uteri were analyzed by HPSEC on an Altex TSK-3000 SW column (600 mm X 7.5 mm I.D.) either before or after incubation with 10 nM [3H]estradiol-17 beta. Postcolumn detection of previously unliganded receptor was accomplished by incubation of fractions (0.38 ml) with 10 nM [3H]estradiol-17 beta for 2-18 h at 0 degrees C. Receptor-bound steroid was separated from free steroid by incubation with small pellets of hydroxylapatite. Nonspecific binding of [3H]estradiol-17 beta in parallel fractions was estimated using an unlabelled competitor (diethylstilbestrol) specific for the estrogen receptor. In low ionic strengths the receptor exists as a single, relatively stable, large form (retention time 34 min). The elution properties of this receptor configuration do not depend on bound steroid ligand. Analysis of receptor at elevated ionic strengths in the presence and absence of steroid ligand suggests that the salt-induced dissociation of receptor components to smaller forms (retention time 47 min) may be partially steroid-dependent. Characterization of receptor in 6 M urea demonstrates the presence of intermediate-sized receptor components (retention time 36-38 min). Analyses of receptor in 6 M urea-0.4 M potassium chloride suggests an inhibition of the more extensive salt-induced dissociation event seen in 0.4 M potassium chloride alone. Furthermore, the intermediate-sized receptor forms seen under these conditions (retention time 41-42 min) are generated in a steroid-dependent manner. The preparation of different molecular forms of biologically active, unliganded estrogen receptor by HPSEC should help further our investigations into the molecular mechanism(s) by which steroid hormones exert their receptor-mediated effects on target cells.

High-performance chromatofocusing of steroid receptor proteins in the presence and absence of steroid. Investigation of steroid-dependent alterations in surface charge heterogeneity.

We have previously reported the development of high-performance chromatofocusing (HPCF) systems for rapid evaluation of the surface charge heterogeneity of steroid receptor proteins, each in the presence of its specific steroid ligand. However, the surface charge properties of ligand-free receptor proteins remain largely unknown. We have now employed HPCF to rapidly evaluate the surface charge properties of cytosolic estrogen receptor proteins in both the presence and absence of the ligand ([3H]estradiol-17 beta). All operations were performed at 0-4 degrees C. Cytosols prepared from immature calf uteri were preparatively analyzed by HPCF on a SynChropak AX-500 column (25 cm X 4.6 mm I.D.) either before or after incubation with 5-10 nM [3H]estradiol-17 beta. Elution of receptor was by generation of internal pH gradients (pH 8.1 to 3.2) using Pharmacia Polybuffers 96 and 74. Postcolumn detection of previously unliganded receptor was accomplished by incubation of pH-neutralized (pH 7.4) fractions with 5 nM [3H]estradiol-17 beta in the presence and absence of unlabelled competitor. Specifically bound steroid was determined in each fraction using an hydroxylapatite adsorption assay. Significant surface charge heterogeneity was observed for both unliganded receptor and the steroid-receptor complex. The heterodisperse pattern of receptor surface charge appeared to vary in a steroid-dependent manner. Preformed steroid-receptor complexes eluted primarily between pH 6.5-7 and between pH 5-6, with indications for heterogeneity within both regions. The surface charge distribution of unliganded receptor routinely revealed additional, more acidic eluting (pH 3.8-4.6) receptor forms. Sodium molybdate, a commonly used receptor-stabilizing agent, maintains receptors during HPCF as relatively acidic eluting forms (pH 3.8-5.0). The specific elution profile of molybdate-stabilized receptor also appears steroid-dependent. These data demonstrate that HPCF can be used preparatively to rapidly isolate unliganded receptor forms in a biologically active state.

Relation between testosterone concentration, sex role identity, and personality among females.

Eighty-four undergraduate female students completed Baucom's Masculinity and Femininity Scales, the Bem Sex Role Inventory, and the Adjective Check List. Testosterone concentration was determined from saliva samples provided by the women. The results indicated that sex role type was related to level of testosterone concentration. More specifically, undifferentiated females had much higher levels of testosterone concentration than did the feminine-sex-typed females. Also, females with high levels of masculinity (androgynous and masculine-sex-typed females combined) had somewhat higher testosterone levels than did feminine-sex-typed females. Adjectival correlates indicated that females with higher testosterone concentrations perceive themselves as self-directed, action-oriented, resourceful individuals; women with lower testosterone concentration view themselves as conventional, socialized individuals, possessing a caring attitude coupled with an anxious and dejected mood.

High-performance chromatofocusing and size-exclusion chromatography: separation of human uterine estrogen-binding proteins.

We have employed high-performance chromatofocusing (HPCF) and high-performance size exclusion chromatography (HPSEC) to separate and identify radiolabelled estrogen binding proteins present in human uterine cytosol. Results obtained using these high-performance methods are compared to results of similar analyses by conventional isoelectric focusing procedures and open-column size exclusion chromatography. By HPCF, descending pH gradients (pH 8-4) allow us to discern four to five estrogen binding proteins with elution pH values typically between pH 4.5 and 7.2. However, when using HPCF, significant quantities of estrogen binding proteins are rarely detected between pH 7.2 and 8.0. This observation has been confirmed by open-column chromatofocusing of these proteins on Polybuffer Exchanger 94. In contrast, preparative isoelectric focusing by electrophoresis in polyampholytes reveals substantial quantities of estrogen binding activity eluted between pH 7.5 and 8.0. Several possible explanations for this disparity are discussed. Apparent differences are also observed when the size heterogeneity of estrogen binding proteins is analyzed simultaneously by size exclusion chromatography in open-column (Sephacryl S-300) and high-performance (TSK-3000SW) modes. HPSEC of these estrogen binding proteins on TSK-3000SW columns demonstrates a predominant 80-85 A species, whereas size exclusion chromatography on conventional Sephacryl S-300 columns reveals two to three distinct regions of estrogen binding proteins with Stokes radii of ca. 85, 60 and 30 A (major species). The larger form of receptor, whether a non-specific aggregate or a multisubunit complex, is stable in unfractionated cytosol and becomes more labile only during size exclusion chromatography.

Transplantation of normal and ectopic human endometrial tissue into athymic nude mice.

Implants or tiny circumscribed nodules of endometrial tissue were found in all female mice given intraperitoneal injections of fragments of human normal (proliferative and secretory) or ectopic (endometrioma) endometrium. Half of these animals received estrogen supplementation and the other half received none. The endometriosis tissue present in these animals at 28 or 56 days after inoculation consisted of glands and stroma with an infiltration of hemosiderin-laden macrophages. Glands in tissue transplants of animals given supplemental estrogen tended to be larger, and the secretory endometrium tended to revert to a proliferative pattern. Palpable nodules at the site of subcutaneous inoculations of proliferative endometrium became undetectable grossly and microscopically within 24 to 32 days, whereas endometrioma tissue remained detectable for up to 70 days and resembled the intraperitoneal tissue microscopically. This study demonstrates that human endometrial tissue can be successfully transplanted into the nude mouse and will retain its basic morphology.

The recovery of normal plasma progesterone levels in the postpartum female.

The return of fertility in postpartum women is gradual. This may be related to ovarian hormonal deficiencies, in particular, inadequate stimulation of the endometrium by progesterone (P). The purpose of this study was to evaluate the recovery of normal luteal function in four postpartum women who were followed for four cycles with serum P, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and prolactin (PRL) taken three times weekly. PRL decreased immediately after delivery and was at normal levels before the first cyclic increases in serum LH and P. E2 and FSH cyclic changes were identical over the four menstrual cycles. LH peak values rose consistently from the first to the fourth cycle. P increased statistically each subsequent cycle in all parameters studied. These parameters of P were peak levels, days to the peak, duration of the luteal phase, and area under the P curve. The postpartum woman thus seems an excellent model for the study of ovarian hormonal disorders, particularly as they relate to disordered luteal P.

Evidence of a possible direct action of Danazol on the human ovary.

Strong evidence is presented that Danazol might exert a direct action on the human ovary by binding to receptor-like receptors for progesterone and testosterone, in concentrations well within the pharmacological range of the treated patient. The possible significance of this finding in understanding Danazol's mode of action is discussed.

PIP: The purpose of this study was to assess the ability of Danazol to bind to high affinity binders for progesterone and testosterone in the cytosol of the human ovary in vitro. Ovaries from women ranging in age from 28-40 years were prepared and used in competition analyses which demonstrated the ability of Danazol to displace testosterone as well as dihydrotestosterone (DHT), from the androgen high affinity binders, and to displace progesterone and R5020, a synthetic compound with high affinity for progesterone receptors, from the progesterone high affinity binder. Use of the recommended dose of 800 mg/24 hours of Danazol for conditions such as endometriosis can lead to significant displacement of progesterone and testosterone by Danazol, assuming that the high affinity binders are not in direct contact with follicular fluid containing much higher levels of progesterone and androgens. Examination of the ovary of a patient treated with Danazol 800 mg/day for 1 year prior to oophorectomy disclosed both the high affinity binder for androgens and the high affinity binder for progesterone that were found in the ovarian cystosol of nontreated patients, proving that longterm treatment with Danazol did not abolish the high affinity binders for either steroid and that a direct action of Danazol at the ovarian level is conceivable. The progesterone and androgen high affinity binders may play a role in ovarian function, hormone production, and ovulation. Danazol binding to them would probably interfere with their normal functioning.