RESUMO
A gonadotropin similar to human chorionic gonadotropin or luteinizing hormone has been demonstrated in rabbit blastocyst prior to implantation. The gonadotropin has been detected by a radioreceptor assay for human chorionic gonadotropin with the use of the plasma membranes of bovine corpora lutea obtained during the first trimester of pregnancy. The concentrations of the human chorionic gonadotropin or luteinizing hormone per milliliter of blastocyst fluid were tenfold higher than those in the blood of pregnant rabbits on days 5 and 6 after mating.
Assuntos
Blastocisto/metabolismo , Implantação do Embrião , Gonadotropinas/biossíntese , Animais , Ligação Competitiva , Blastocisto/análise , Membrana Celular/metabolismo , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/metabolismo , Corpo Lúteo/metabolismo , Feminino , Gonadotropinas/análise , Humanos , Radioisótopos do Iodo , Hormônio Luteinizante/sangue , Hormônio Luteinizante/metabolismo , Gravidez , Testes de Gravidez , Ligação Proteica , Coelhos , Ensaio RadioliganteRESUMO
A group of 590 women who, 4 to 5 weeks after their last menstrual period, were confirmed to be pregnant, as measured by the human chorionic gonadotropin (hCG) by radioreceptor assay. Nine of these women had serum hCG levels approximately twofold higher than the others and were suspected of having twin pregnancy. When these women were tested at 12 weeks of gestation, pelvic sonography confirmed twin pregnancies in all the nine cases. Serum hCG levels thus provide a simple, rapid, and easy method to detect twin pregnancy.
Assuntos
Gonadotropina Coriônica/sangue , Testes Imunológicos de Gravidez , Gravidez Múltipla , Feminino , Humanos , Gravidez , Primeiro Trimestre da Gravidez , GêmeosRESUMO
A rapid, sensitive, and specific radioreceptor assay for the determination of human chorionic gonadotropin and luteinizing hormone in plasma is described. Plasma membranes of bovine corpora lutea of early pregnancy, which bind biologically active labeled human chorionic gonadotropin, have been used as receptor. Pregnancy could be detected by assaying the gonadotropin in plasma samples obtained from day 6 to 8 after conception.
Assuntos
Gonadotropina Coriônica/sangue , Testes de Gravidez , Ensaio Radioligante , Acromegalia/sangue , Animais , Bovinos , Corpo Lúteo/metabolismo , Feminino , Humanos , Hipotireoidismo/sangue , Lactação , Hormônio Luteinizante/sangue , Menstruação , Gravidez , Receptores de Superfície Celular , Fatores de TempoRESUMO
Based on the regeneration of the hormonal activity following recombination, the alpha and beta subunits of human follicle-stimulating hormone have been designated as 'functional' or 'nonfunctional'. Chemical modifications of the tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone, luteinizing hormone, and the 'functional' human follicle-stimulating hormone alpha and beta subunits have indicated that the tryptophan in human follicle-stimulating hormone-beta and human luteinizing hormone-beta is essential for the biological activity. The iodination of human follicle-stimulating hormone-alpha did not interfere with the hormonal activity. The modification of arginine abolishes the biological activity of the hormones. The accessibility of tyrosine and methionine in human follicle-stimulating hormone-alpha, of arginine in both native hormones and subunits, and the non-availability of the tryptophan residues to 2-hydroxy 5-nitrobenzyl bromide suggest that the alpha subunit lies on the surface of the native molecule.
Assuntos
Arginina , Hormônio Foliculoestimulante , Metionina , Triptofano , Tirosina , Sequência de Aminoácidos , Brometo de Cianogênio , Glioxal/análogos & derivados , Hormônio Liberador de Gonadotropina , Humanos , Iodetos , Iodoacetatos , Hormônio Luteinizante , Substâncias MacromolecularesRESUMO
Human follicle-stimulating hormone (human FSH), dansylated human FSH, human FSH-alpha and human FSH-beta subunits were individually conjugated by photoactivation to an azidobenzoyl derivative of a glycopeptide isolated from fetuin. The conjugates were purified on a column of Sephadex G-100. The carbohydrate content of the conjugated human FSH increased 2.7-fold with a concomitant 1.5- and 2-fold increase in the immunological and biological activity of the human FSH. The glucosamine content of the human FSH-alpha and human FSH-beta increased 6- and 1.4-fold, respectively, after conjugation. Human FSH-alpha conjugate, when recombined with untreated human FSH-beta showed up to 50% increase in the biological activity over the control. When the conjugated human FSH-beta was combined with untreated human FSH-alpha, there was little change in the biological activity. These experiments demonstrate that the photoactivation procedure, although random and site-nonspecific in nature, provides a potential means of attachment of glycopeptides to the protein moiety and enhancement of the hormonal activity of human FSH.
Assuntos
Hormônio Foliculoestimulante , alfa-Fetoproteínas , Configuração de Carboidratos , Sequência de Carboidratos , Humanos , Substâncias Macromoleculares , Fotoquímica , Ligação ProteicaRESUMO
Human follicle-stimulating hormone (FSH) was digested with subtilisin, thermolysin, cyanogen gromide, pronase and trypsin to isolate the cystine-containing peptides. These peptides were purified by gel filtration through Sephadex G-50 column and by high-voltage paper electrophoresis at pH 6, 3.5 and/or 2. The location of the cystine-containing peptides in human FSH alpha- and beta-subunits was established by amino acid composition, end-group analysis and determination of the amino acid sequence by Edman degradation. The results indicate that the disulfide bonds are present between half-cystine residues located between positions 7 and 10, 28 and 87 and 82 and 84 in the alpha-subunit, and between positions 3 and 28, 17 and 51 and 32 and 104 in the beta-subunit of human FSH.
Assuntos
Dissulfetos/análise , Hormônio Foliculoestimulante , Hipófise/análise , Animais , Bovinos , Brometo de Cianogênio , Cistina/análise , Humanos , Fragmentos de Peptídeos/análise , Conformação Proteica , Ovinos , Especificidade da Espécie , Subtilisinas , Suínos , TermolisinaRESUMO
Amniotic fluid, obtained from women at term, was centrifuged and concentrated by ultrafiltration. The concentrated amniotic fluid was purified by gel filtration on Sephadex G-100 and ion-exchanged chromatography on DEAE-cellulose. The fractions obtained during purification procedures were assayed for prolactin and somatotropin activities by respective radioimmunoassays. The prolactin-rich fraction obtained from ion-exchange chromatography was further purified by isoelectric focusing. A yield of 0.21 mg of highly pruified prolactin containing 40 units/mg was obtained from 1 liter of the amniotic fluid. The prolactin was free of somatotropin and human placental lactogen.
Assuntos
Líquido Amniótico/análise , Prolactina/isolamento & purificação , Feminino , Hormônio do Crescimento/análise , Humanos , Lactogênio Placentário/análise , Gravidez , RadioimunoensaioRESUMO
An improved method is described for the purification of prolactin from the same batch of acetone-dried pituitary glands which is used for the isolation of other anterior pituitary hormones. The residue obtained after the extraction of the glycoprotein fraction from the pituitary acetone powder was extracted for growth hormone. The supernatant after the precipitation of growth hormone at 50% ethanol concentration was adjusted to 85% in ethanol. To the supernatant obtained from that step an equal volume of acetone was added to precipitate the 'PRL fraction.' This fraction was further purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose and isoelectric focussing. A yield of 23 mg prolactin, 37 U/mg, was obtained per 1000 g pituitary acetone powder.
Assuntos
Hipófise/análise , Prolactina/isolamento & purificação , Acetona , Cromatografia em Gel , Eletroforese Descontínua , Humanos , Focalização Isoelétrica , MétodosRESUMO
The use of specific receptors has provided a novel method to prepare LH-free serum. LH-free serum was used in RRA to measure basal LH levels in serum of men and women during menstrual cycle as well as during post menopause.
Assuntos
Hormônio Luteinizante/sangue , Ensaio Radioligante , Receptores de Superfície Celular , Membrana Celular/metabolismo , Corpo Lúteo/metabolismo , Feminino , Humanos , Masculino , Menopausa , Menstruação , Métodos , Gravidez , Radioimunoensaio , Receptores de Superfície Celular/metabolismoRESUMO
A simple, specific, rapid, centrifugation-free solid phase RIA for hCG has been developed. Purified gamma-globulin isolated from antisera produced against hCG-beta in rabbits is covalently linked to glutaraldehyde-activated aminopropyl glass beads. The entire assay can be completed within 3 h at room temperature. The sensitivity (1 ng/ml), precision (intra- and interassay variabilities, less than 10% and less than 15%, respectively), and specificity (0.4% cross-reactivity with LH) are comparable to other methods. The hCG levels determined by the present method show good correlation with those obtained by another RIA and by radioreceptorassay. The present method also provides the basis for the development of similar rapid and sensitive assays of other hormones.
Assuntos
Gonadotropina Coriônica/análise , Reações Cruzadas , Feminino , Vidro , Humanos , Microquímica , Gravidez , Radioimunoensaio/métodosRESUMO
We describe a patient with onset of puberty at the age of 5 yr. characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins.
Assuntos
Mutação , Puberdade Precoce/genética , Receptores do LH/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Criança , Gonadotropina Coriônica/farmacologia , AMP Cíclico/biossíntese , DNA/análise , Éxons , Humanos , Masculino , Reação em Cadeia da Polimerase , Puberdade Precoce/tratamento farmacológico , Receptores do LH/química , Receptores do LH/metabolismo , Análise de Sequência , Homologia de Sequência , Transfecção , Pamoato de Triptorrelina/uso terapêuticoRESUMO
This study was designed to test the feasibility of a patient-monitored glucose determination program to establish and maintain normal blood glucose levels. Ten pregnant women, who were insulin-dependent diabetics prior to becoming pregnant and who were in their eighth week or less of pregnancy, were offered the program. All 10 accepted and continued the program for the duration of their pregnancy. Normal plasma glucose (60 to 140; mean = 80 mg/dl) levels were achieved after one week of the program and were maintained throughout the pregnancy as documented by 5 to 8 blood glucose determinations a day. The hemoglobin A1c level, which was elevated in all 10 patients at the start (9.4 +/- 1.6 per cent) of the program, fell into the normal range (2 to 5.0 per cent) five weeks after glucose values became normal. Serum estradiol (0.8 +/- 0.6 ng/ml), serum prolactin (10 +/- 9 ng/ml) and serum human chorionic gonadotropin (5,500 +/- 1,700 ng/ml), although all abnormal at the start of the program, became normal after glucose control was achieved (program weeks 4, 5 and 6, respectively). The infants showed no signs of macrosomnia (2,988 +/- 959 g), hypoglycemia, hyperbilirubinemia, hypocalcemia, erythremia or respiratory distress. Therefore, a program to maintain normal blood glucose levels during a diabetic patient's pregnancy is not only possible but may also improve the pregnancy and the outcome.
Assuntos
Glicemia/metabolismo , Insulina/uso terapêutico , Gravidez em Diabéticas/sangue , Adulto , Glicemia/análise , Gonadotropina Coriônica/sangue , Dieta para Diabéticos , Estradiol/sangue , Feminino , Hemoglobina A/análise , Humanos , Cooperação do Paciente , Gravidez , Gravidez em Diabéticas/terapia , Prolactina/sangueRESUMO
A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concentrated by ultrafiltration. The filtrate, containing < 50,000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) activity. The filtrate was ultrafiltered sequentially through Amicon PM-10, PM-30 and UM-2 filters to yield a LH-RBI-containing fraction in the higher molecular weight range of 30,000-10,000 and a LH-RBI-containing fraction in the lower molecular weight range of 10,000-1000. The higher molecular weight LH-RBI fraction was purified on Sephadex G-25 and the lower molecular weight LH-RBI fraction was purified on Sephadex G-50. Both the high- and the low-molecular-weight LH-RBI species inhibited the binding of 125I-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Similarly, the production of testosterone by hCG-stimulated rat Leydig cells was inhibited in a dose-response manner by both the high- and the low-molecular-weight LH-RBI species. The LH-RBI activity in the low-molecular-weight species was stable at 4 degrees C for up to 6 months and at temperatures up to 90 degrees C for 15 mins, whereas the LH-RBI activity of the high-molecular-weight species was stable at 4 degrees C for 15 months and unstable at 60 degrees C after 15 min. The 7000 g supernatant provided a much-needed source to obtain larger than previously reported quantities of LH-RBI for isolation as well as for structure and function studies.
Assuntos
Corpo Lúteo/metabolismo , Peptídeos/análise , Receptores do LH/antagonistas & inibidores , Animais , Bovinos , Gonadotropina Coriônica/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Células Intersticiais do Testículo/metabolismo , Masculino , Peso Molecular , Peptídeos/isolamento & purificação , Ligação Proteica , Ensaio Radioligante , Ratos , Receptores do LH/isolamento & purificaçãoRESUMO
Four baboons (Papio anubis) were actively immunized with bovine LH receptor for periods of 6-22 months. Serum antibody levels were measured by an enzyme immunoabsorbant assay (ELISA). Antibodies against the receptor were detected 2 weeks after the first injection. Antisera caused an inhibition in the binding of human chorionic gonadotropin (hCG) to its receptor as well as inhibited the production of hCG induced testosterone by rat Leydig cells in culture. Serum estradiol and progesterone levels were determined by radioimmunoassay (RIA). Progesterone levels were suppressed during the post immunization period. Two baboons experienced periods of anovulation. Serum estradiol levels were cyclic and appeared elevated. Baboons were mated with males of proven fertility; none of the immunized females conceived over eight cycles of observation. Fertility parameters returned to normal, when antibodies against LH-receptor were undetectable in the serum.
Assuntos
Anticorpos/fisiologia , Fertilidade/imunologia , Receptores do LH/imunologia , Animais , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Estradiol/sangue , Feminino , Imunização , Células Intersticiais do Testículo/metabolismo , Masculino , Papio , Progesterona/sangue , Receptores da Gonadotropina/metabolismo , Testosterona/biossínteseRESUMO
Monoclonal antibodies (Mabs) were produced against bovine Lutropin receptor (LH-R). Antibodies were detected by an enzyme linked immunosorbant assay (ELISA). Hybridomas were subcloned to achieve monoclonality. Ascites were developed in Balb/c mice. Hybridoma supernatants were purified by ammonium sulfate precipitation and chromatography on hydroxylapatite columns. LH-R antibodies showed upto 50% inhibition of 125I-labeled hCG binding to bovine luteal cell membranes and up to 80% inhibition of testosterone production by hCG stimulated mouse Leydig cells. LH-R antibodies were predominantly IgM isotype. Purified antibodies showed a 78-kDa band, in SDS-PAGE, as the heavy chain of the immunoglobulin. LH-R antibodies were localized specifically in the thecal and luteal cells of the rat ovaries as well as in the Leydig cell of mouse testes. Injections of the LH-R antibody caused a constant estrus in normal rats. One month after the cessation of the injections the animals returned to normal estrus cycle and fertility. Pregnant mice injected with LH-R antibodies produced only 3 viable pregnancies and 10 pups, as compared to 8 pregnancies and 45 pups born to normal controls. LH-R antibodies also caused, approximately, a 50% reduction in testosterone production in normal male rats. These observations indicate a high degree of specificity of the Mab to LH-R and their potential in studies on gonadal function.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores do LH/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Bovinos , Cromatografia por Troca Iônica , Corpo Lúteo/metabolismo , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Hibridomas/imunologia , Isotipos de Imunoglobulinas/análise , Fosfatos de Inositol/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovário/metabolismo , Ratos , Receptores do LH/biossíntese , Transdução de Sinais , Testículo/imunologiaRESUMO
Serum testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay in 14 patients with unaborted hydatidiform mole and in 16 patients with normal pregnancy of similar gestational age. Serum human chorionic gonadotropin (hCG) was measured by the radioreceptor assay in patients with hydatidiform mole. Serum T ranged from 0.27 to 5.39 ng/ml with a mean +/- SE of 2.21 +/- 0.45 ng/ml in patients with hydatidiform mole mole and from 0.20 to 2.40 ng/ml with a mean +/- SE of 0.80 +/- 0.14 ng/ml in patients with normal pregnancy, the difference being statistically significant (P = less than 0.005). Similarly, patients with molar pregnancies had a significantly higher (P = less than 0.005) serum DHT (range: 0.09 to 0.62 ng/ml; mean +/- SE: 0.29 +/- 0.05 ng/ml) than patients with normal pregnancies (range: 0.04 to 0.28 ng/ml; mean +/- SE 0.12 +/- 0.02 ng/ml). There was no significant correlation between uterine size or serum hCG and serum T or DHT. The possible sources of the elevated serum T and DHT and the lack of hirsutism or virilization in patients with trophoblastic disease are discussed.
Assuntos
Di-Hidrotestosterona/sangue , Testosterona/sangue , Neoplasias Trofoblásticas/sangue , Neoplasias Uterinas/sangue , Feminino , Humanos , Mola Hidatiforme/sangue , GravidezRESUMO
Plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH), prolactin (PRL), estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) were measured by radioimmunoassay in 7 patients who had ovulation induction with clomiphene citrate and human chorionic gonadotropin. Six of the 7 patients ovulated. There was higher increase in FSH than LH in 3 patients who also showed a midcycle FSH and LH peak. Plasma PRL was initially elevated in 2 patients, remained normal (less than 25 ng/ml) throughout the cycle in one patient, and showed an increase in the late luteal phase in 3 patients. Plasma E2 showed a good response at the end of clomiphene intake, with a subsequent preovulatory surge. Plasma P increased to more than 25 ng/ml in patients who ovulated and reached higher levels than in normal ovulatory cycles. Plasma T increased and decreased in a similar pattern to plasma E2 and was higher with clomiphene therapy than in the normal menstrual cycle. Plasma DHT remained similar to concentrations found in normal menstrual cycles. The findings are discussed in relation to the effect of clomiphene and these hormones on the pituitary-ovarian axis, ovulation rate, and conception rate.
Assuntos
Clomifeno/uso terapêutico , Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Indução da Ovulação , Prolactina/sangue , Gonadotropina Coriônica/uso terapêutico , Di-Hidrotestosterona/sangue , Estradiol/sangue , Feminino , Humanos , Progesterona/sangue , Radioimunoensaio , Testosterona/sangueRESUMO
Serum human chorionic gonadotropin (hCG) was measured by a radioreceptorassay (RRA) and radioimmunoassay (RIA) and serum hCG-beta and hCG-alpha by RIA in 10 patients with intact mole, 3 patients with choriocarcinoma, and 4 patients with hydatidiform mole during treatment. hCG levels by RRA were higher in 5 of 10 molar pregnancies and ranged from 20,900 to 100,000 ng/ml and from 30,000 to 100,000 ng/ml by RIA. hCG levels by RRA and RIA paralleled one another closely during treatment of hydatidiform mole. hCG-alpha was higher than hCG by RRA and RIA and hCG-beta in molar pregnancies, in the uterine venous blood draining a uterine choriocarcinoma, and during chemotherapy of choriocarcinoma. In 2 of 3 choriocarcinoma patients who eventually developed cerebral metastases, hCG-alpha increased while hCG and hCG-beta were declining or negative. hCG-beta was usually lower than hCG or hCG-alpha in all the cases studied. These results demonstrate the production of free alpha and beta subunits in trophoblastic disease. Further, due to the biospecificity, simplicity, and rapidity, the RRA of hCG is a sueful diagnostic aid during treatment of trophoblastic neoplasia until the levels fall to within the sensitivity range of the assay. Finally, the RIA of hCG, hCG-beta, and hCG-alpha, which requires several days, should be performed until they become negative or fall within normal range.
Assuntos
Coriocarcinoma/metabolismo , Gonadotropina Coriônica/metabolismo , Mola Hidatiforme/metabolismo , Neoplasias Uterinas/metabolismo , Feminino , Humanos , Radioisótopos do Iodo , Hormônio Luteinizante/metabolismo , Gravidez , Radioimunoensaio , Ensaio Radioligante , Neoplasias Trofoblásticas/metabolismoRESUMO
Nonradioisotopic enzyme assays have been developed to detect the preovulatory luteinizing hormone (LH) surge in the unextracted urine of normally menstruating women. The amino groups on 6-mm glass beads were activated and coupled to the anti-LH-gamma-globulin for enzyme immunoassay or to the LH receptor prepared from bovine corpora lutea for enzyme receptorassay. The LH was conjugated with alkaline phosphatase. The enzyme assays detected preovulatory LH surge in the morning urine samples of 12 normally menstruating women. The LH surge was coincidental with preovulatory serum estradiol rise, shift in basal body temperature, and 4+ spinnbarkeit of the cervical mucus. The serum estradiol and progesterone levels in the luteal phase were indicative of corpus luteum formation.
Assuntos
Técnicas Imunoenzimáticas , Hormônio Luteinizante/urina , Ovulação , Adulto , Fosfatase Alcalina , Temperatura Corporal , Muco do Colo Uterino , Estradiol/análise , Feminino , Humanos , Hormônio Luteinizante/metabolismo , Radioimunoensaio , Receptores de Superfície Celular/análise , Receptores do LHRESUMO
The authors report the case of a 23-year-old woman with a virilizing lipoid cell tumor of the ovary. The patient developed precocious puberty at age 4 and from age 5 to 10 years was treated weekly with intramuscular medroxyprogesterone acetate. She began to menstruate spontaneously at age 13, but developed hirsutism, acne, and irregular menses at age 15 followed by secondary amenorrhea at age 20. Steroid analyses demonstrated elevated peripheral plasma levels of testosterone, dihydrotestosterone, androstenedione, 17 alpha-hydroxyprogesterone, progesterone, desoxycorticosterone, estrone, and estradiol. Catheterization of the adrenal and ovarian veins revealed increased secretion of testosterone, dehydroepiandrosterone, androstenedione, 17 alpha-hydroxyprogesterone, progesterone, desoxycortcosterone, and corticosterone, 11-desoxycortisol, cortisol, estrone, and estradiol from the right ovarian vein. At surgery a right ovarian lipoid cell tumor was removed. Incubation studies with the tumor tissue confirmed the presence of gonadal and adrenocortical steroids. The tissue also contained high-affinity human chorionic gonadotropin-luteinizing hormone binding sites with normal binding affinity.