Inhibition of gonadotropin by delta9-tetrahydrocannabinol:mediation by steroid receptors?

Competition assays for estradiol receptors in cytosol preparations of uteri from rhesus monkeys and humans showed that delta9-tetrahydrocannabinol (THC) does not compete with estradiol for intracellular estrogen recptors. Although isotopically labeled THC bound to macromolecules in uterine cytosol from the rhesus monkey, the binding was not displaced by unlabeled THC, diethylstilbestrol, estradiol, progesterone, cortisol, or 5 alpha-dihydrostestosterone. Scatchard analyses indicated that high-affinity saturable binding of THC to cytosol did not occur. Thus the inhibitory effect of THC on gonadotropin and steroid secretion in primates is not mediated by the interaction of THC with intracellular steroid hormone receptors.

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.

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).

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.

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.

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.

Saliva as a matrix for measuring free androgens: comparison with serum androgens in polycystic ovarian disease.

We report a simple and direct procedure for the measurement of circulating free testosterone concentrations by using saliva as a matrix rather than serum. There is a close correlation between saliva testosterone values measured by radioimmunoassay, calculated values of free testosterone, and free testosterone estimated by equilibrium dialysis. Our method is direct and has the advantage that the biologic fluid can be obtained routinely by noninvasive techniques outside the clinic during a course of therapy. We also show that a single saliva value is of greater diagnostic use than any of the currently used androgen assays. Testosterone was found to be elevated in the saliva of 17 infertility patients diagnosed as having polycystic ovarian syndrome, 14 of these patients were hirsute.

The use of synthetic luteinizing hormone-releasing hormone in induction of ovulation.

Each of 14 anovulatory patients received a single injection of 150 micrograms of synthetic luteinizing hormone-releasing hormone (LH-RH) in one induced menstrual cycle. In two subsequent cycles, patients were pretreated with clomiphene before LH-RH injection. Four patients received LH-RH after pretreatment with human menopausal gonadotropin (HMG) in another cycle. In each cycle, gonadotropin release and ovulation were recorded. All patients had previously failed to ovulate when treated with large doses of clomiphene. No patient ovulated following injection of LH-RH alone, although five patients exhibited a good pituitary response. Nine patients ovulated when they received LH-RH after pretreatment with clomiphene, and one patient ovulated when pretreated with HMG. The diagnostic value of a single injection of LH-RH is not clear. In the present study, gonadotropin response to LH-RH was not an entirely accurate predictor of a patient's ovulatory response in any of the four cycles. On the other hand, when clomiphene-LH-RH was administered, a good response was associated with ovulation in that cycle. The exact role of LH-RH in inducing ovulation is unclear, but the results of using LH-RH in conjunction with clomiphene are encouraging enough to warrant continued use and further study.

Prolactin receptors in the ovary.

The binding of prolactin (PRL) to the plasma membranes of bovine and human ovaries was investigated using both homologous and heterologous 125I-prolactin. Saturation and Scatchard analysis demonstrated that human prolactin binds to human ovarian membranes with a Kd of 2 x 10(-10) M; to bovine ovarian membranes with a Kd of 1.9 x 10(-10) M; and to bovine corpora lutea membranes with a Kd of 1.9 x 10(-10) M. The concentrations of binding sites in bovine and human ovaries were 2.9 x 10(-15) moles/mg of protein and 2.0 x 10(-15) moles/mg of protein, respectively. The number of bindings sites in the bovine corpora lutea was 1.5 x 15(-15) moles/mg of protein. Specificity studies with bovine PRL, ovine PRL, human luteinizing hormone, human follicle-stimulating hormone, and bovine growth hormone showed this binding to be specific. Comparison of binding of PRL to membranes of other target and nontarget tissues suggests that the ovary is one of the primary target tissues. These data suggest that prolactin plays a role in the ovarian cycle.

Effect of tetrahydrocannabinol on the hypothalamic-pituitary axis in the ovariectomized rhesus monkey.

Single doses of delta9-tetrahydrocannabinol (THC) (5.0, 2.5, 1.25, or 0.625 mg/kg) can decrease the levels of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the ovariectomized rhesus monkey. The inhibition of gonadotropins (50% to 88%) lasts for 6 to 24 hours depending upon the dose of THC. There are no great differences in the responses of the two gonadotropins to THC. The inhibition of gonadotropin levels by THC appears to be at the level of the hypothalamus, since both LH and FSH are released from the pituitary gland in response to LH- releasing factor in the presence of THC.

Surgical correction of the septate uterus.

PIP: Metroplasty for septate uterus performed in 28 patients at Methodist Hospital in Houston is discussed. The preoperative fetal survival rate was 19.5%. Prior to laparotomy, D and C was performed in all patients. During the operation a lower abdominal transverse incision was used for abdominal exploration. The uterus was first divided from above downward in an anteroposterior direction until the endometrial cavity was entered. The incision was kept in the midline to minimize blood loss. Once the endometrial cavity was exposed, scissors were used to cut the septum bilaterally without excising it. Care was taken not to cut into the myometrium superiorly. The uterine walls were then reapproximated by simple interrupted no. 1 chromic catgut sutures that transversed serosa, myometrium, and endometrium. The first sutures were placed on the anterior and posterior aspects of the uterus just below the incision in the uterine wall. The remaining sutures were placed about .5 to 1 cm apart; none were tied until all sutures were in place. Closure was in a longitudinal anteroposterior direction along the same line as the incision. Approximation of the serosal surface was further enhanced by a running inverting 3-0 chromic catgut suture. In 16 patients, an IUD was placed in the uterine cavity before closure and left in place for 1 to 3 months. The frequency of associated endometriosis was 32.1%. Of the patients wishing to conceive, 73% became pregnant. The postoperative fetal survival rate was 87.5%. Delivery was by cesarean section.^ieng

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.

Practical uses of steroids and gonadotropins in obstetrics and gynecology.

PIP: Several steroid and gonadotropin determinations that are available to practicing physicians are discussed and their practical uses are emphasized. Before undertaking a workup of a patient who appears to have an endocrine abnormality a physician should know the availability and benefits of such tests, the time and expense involved, and the interpretation of laboratory reports. Estrogens are phenolic steroids secreted by the ovaries, adrenal glands, testicles, and the fetal-placental unit and are found in over 20 metabolites in the urine; the most important are estrone, estridiol, and estriol. Total or fractional determinations of urinary estrogens are expensive and may require 7 days. Human gonadotropin determinations are expensive and require 2 weeks. Contraceptive medications, tranquilizers, and sedatives interfere with secretion of this hormone. Clinical usefulness is of value only when dterminations are extremely high or repeatedly ver y low. Culdoscopic findings may be of more value than either estrogen or gonadotrpin determination. Estrogen determinations can be of value in male patients with gynecomastia, in suspected fetal distress or fetal death in utero (a drop of 50% in the normal 1000-fold increase of pregnancy indicates need for delivery of infant), and when an indication of size of infant is needed to determine elective repeat ceasarean sectton. Ther is no practical value of pregnanediol determination in pregnancy or in the amenorrheic female. The clinical use of 17-hydroxycorticoids is related to its value as a screening procedure for adrenal disorders. Patient stress and several drugs may interfere with accurate tests. The 17-ketosteroids are used primarily for the sam e purpose. Progesterone determinations are useful to differentiate ovarian from adrenal pathology. Urinary testosterone increase may be present in some ovarian tumors. Human chorionic gonadotropin (HCG) detection is used for pregnancy tests; a sensitive technique may obtain a positive test day Day 30 of the menstrual cycle or 16 days after conception. These tests are also used in the diagnosis of hydatidiform mole, choriocarcinoma, and to guide care of trophoblastic disease. Of newer methods the competitive protein binding technique for estrogen, progesterone, and testosterone is rapid, accurate, and sensitive. Radioimmunoassay for FSH, LH, and other trophic hormones is complex but promising. Conversion studies are complex but may be of use.^ieng

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.

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)