Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Synergic Effects of β-Estradiol and Erythromycin on hERG Currents

The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I _Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62% of control with an IC₅₀ of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8% of control with an IC₅₀ of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.     

Novel multialveolar epithelial structures from rabbit mammary gland that synthesize milk specific fatty acids in response to prolactin

Summary Multialveolar mammary epithelial structures have been prepared from rabbit mammary gland by treating the tissue with collagenase plus hyaluronidase. These structures synthesize milk specific fatty acids when cultured with physiological concentrations (0.05 μg/ml) of prolactin in the presence of insulin and corticosterone. They have many of the advantages but few of the disadvantages of either mammary explains or primary cells in culture. For example, they are easily prepared in large numbers and respond to prolactin in culture even in the absence or other tissue extracts. Because their level of organization is intermediate between that of explants and single cells, they provide a complementary system for studies on mammary differentiation. This work was supported by grants from the Agricultural Research Council of Great Britain and the Royal Society to R. R. Dils, by U.S. Public Health Service Grant CA-16392, and an American Cancer Society-Eleanor Roosevelt International Cancer Fellowship to H. L. Hosick. Prolactin was a gift from the Endocrinology Study Section, National Institutes of Health, Bethesda, MD.     

Comparable Attenuation of Aβ<Subscript>25–35</Subscript>-Induced Neurotoxicity by Quercitrin and 17β-Estradiol in Cultured Rat Hippocampal Neurons

In the present work, potential protective effects of quercitrin (a phytoestrogen) on Aβ-induced neurotoxicity in cultured rat hippocampal neurons were investigated in comparison with 17β-estradiol. Cell viability, oxidative status, and antioxidative potentials were used as comparative parameters. Co-exposure of cultured neurons to Aβ_25–35 with either quercitrin or 17β-estradiol (50–100 μM) for 72 h attenuated Aβ_25–35-induced neurotoxicity and lipid peroxidation, but not Aβ_25–35-induced ROS accumulation. However, only 17β-estradiol counteracted a reduction in glutathione content and only quercitrin counteracted a reduction in glutathione peroxidase activity. Both compounds displayed no effects on superoxide dismutase activity. A specific estrogen receptor antagonist, ICI 182780, did not abolish neuroprotective effects of quercitrin and 17β-estradiol. These findings suggested that quercitrin and 17β-estradiol attenuated Aβ_25–35-induced neurotoxicity in a comparable manner. Underlying neuroprotective mechanisms of both compounds were probably not related to estrogen receptor-mediated genomic mechanisms but might involve with their antioxidant and free radical scavenging properties.     

Histochemical studies of rat ovarian follicular cells in vitro

Summary Membrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δ^p5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation. This research was supported in part by a Faculty Research Grant from the Horace H. Rackham School of Graduate Studies, and by United States Public Health Service Grants RR-05383-09 and AM-06918-06.     

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO₅ medium). Growth in WO₅ medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO₁₆₀ medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO₅ medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×10⁷ plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO₅ medium. This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation.     

Changes of Sodium Iodide Symporter Regulated by IGF-I and TGF-β1 in Mammary Gland Cells from Lactating Mice at Different Iodine Levels

The sodium/iodide symporter (SLC5A5, also known as NIS) is a transmembrane glycoprotein. Physiologically, iodide transportation in the mammary gland occurs during late pregnancy and lactation. To identify factors that may regulate this process at different iodine levels, we have studied the expression of NIS gene and protein in cultured mammary gland explants from lactating mice by real-time quantitative PCR and In-Cell Western methods. Mammary gland cells were grown in media with different levels of iodine for 24 h. The iodine treatment groups consist of low iodine group I (LI-I, 0 μg/l), low iodine group II (LI-II, 5 μg/l), control group (C, 50 μg/l), high iodine group I (HI-I, 3,000 μg/l), and high iodine group II (HI-II, 10,000 μg/l). The cells were then incubated with or without insulin-like growth factor I (IGF-I) or transforming growth factor β1 (TGF-β1) for another 24 h. We found that iodine inhibited NIS mRNA and protein expression in a dose-dependent manner. IGF-I and TGF-β1 further decreased NIS mRNA and protein expression that iodine inhibited at different iodine levels. In summary, we have shown that iodine downregulated NIS expression in cultured mammary gland explants from the lactating mouse. IGF-I and TGF-β1 inhibited NIS mRNA and protein expression in the mammary gland under different iodine levels.     

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland

Summary Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 μg/ml), aldosterone (1 μg/ml), growth hormone (5 μg/ml), cortisol (5 μg/ml), and prolactin (80 ng/ml, present as a contaminant in 5 μg/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNA_csn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo. This work was supported by Department of Health, Education and Welfare Grants CA11058 and CA25304 from the National Cancer Institute.     

Whole mammary gland organ culture: Selection of appropriate gland

Summary This report presents the results of studies on differences in the responsiveness of the different mammary glands of virgin mice in whole mammary gland organ culture. The entire second and third thoracic and the fourth inguinal mammary fat pads containing the parenchyma were excised and incubated in Waymouth's medium (MB752/1) supplemented with the hormones estradiol, progesterone, aldosterone, insulin, growth hormone, and prolactin. The rate of DNA synthesis was determined by acid-insoluble [³H]-thymidine radioactivity. Morphological measures of the extent of lobulo-alveolar development in the parenchyma were used as criteria of induction of mammogenesis in organ culture. It was evident that, after 3 and 5 days incubation in vitro, the mammary parenchyma in the second thoracic fat pad is the most responsive to the hormone-supplemented medium. This research was supported by United States Public Health Service Grant CA-11058 and Contract E-72-3212 from the National Cancer Institute.     

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49

Three antisera to myelin basic protein—a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP—were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69–84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69–71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69–71. S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.This work supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by NS-15322 from the National Institutes of Health of the U.S. Public Health Service; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Effects of pregnenolone,cyanoketone, and equilenin on the human malignant trophoblast in vitro

Summary Cultures of human malignant trophoblast cells were studied to determine the basis of inhibition of human chorionic gonadotropin (HCG) secretion and depletion of glycogen following incubation of the cells in the presence of pregnenolone (3β-hydroxypregn-5-en-20-one). Incubation of the cells for as long as 3 days with 5 or 10 μg of pregnenolone per ml resulted in decreased protein content, inhibition of DNA synthesis, diminished glucose utilization, and marked accumulation of acellular debris in the medium. These changes became more pronounced with time of incubation and were related to the dose of pregnenolone employed. The effect of pregnenolone on all of the parameters measured was mimicked and potentiated by either equilenin (3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one) or cyanoketone (androst-5-en-2α-cyano-17β-hydroxy-4,4,17α-trimethyl-3-one), inhibitors of pregnenolone conversion to progesterone. These results suggested that the glycogenolysis and inhibition of HCG secretion that occur when the trophblast cells are incubated in the presence of pregnenolone result from toxicity rather than from cellular differentiation, and that prior conversion of pregnenolone to progesterone is not necessary for the manifestation of the toxicity. This work was supported in part by Contract PH 43-NCI-E-68-1010 from the Special Virus Cancer Program, National Cancer Institute, United States Public Health Service.     

Characterization of neuronal zebra finch GABAA receptors: steroid effects

Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize γ-aminobutyric acid (GABA) type A receptors (GABA_A) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation by 17β-estradiol(E₂), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked currents were inhibited by picrotoxin (10 μmol l⁻¹) and bicuculline methiodide (10 μmol l⁻¹) and potentiated by pentobarbital (100 μmol l⁻¹) and propofol (3 μmol l⁻¹). Loreclezole (10 μmol l⁻¹) potentiated GABA-evoked currents, suggesting the presence of β₂, β₃ and/or β₄ subunits. Diazepam (1 μmol l⁻¹) potentiated currents, while Zn²⁺ (1 μmol l⁻¹) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l⁻¹) potentiated currents, whereas E₂ (1 μmol l⁻¹), 5α- and 5β-DHT (1 μmol l⁻¹), and corticosterone (10 μmol l⁻¹) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABA_A receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little or no acute modulation by sex steroids or corticosterone. Accepted: 12 November 1997     

Isolation of cell lines from embryos of the cockroach,Blattella germanica

Summary Cell lines were isolated from three stages of embryos ofBlattella germanica dissociated with trypsin. The lines have been subcultured 50 to 134 times in 3 years. Line UM-BGE-1 was isolated from germ band embryos at stages of segmentation and limb-bud formation (5 days old). Line UM-BGE-2 was derived from embryos at dorsal closure (7 days old). Line UM-BGE-4 arose from embryos in the germ band and dorsal closure stages (5 and 7 days old); these cells colonize as hollow spheres or vesicles. Line UM-BGE-5, isolated during organogenesis (10 days old), developed into two distinct sublines. Subline α is composed of round cells that do not attach to the flask. Subline β grows as an attached monolayer; the cells can be removed with a saline solution containing 20mM disodium dihydrogen Versenate^?. Most of the cells of these lines have the diploid chromosome number (23 or 24) excepting line UM-BGE-1 in which the tetraploid number predominates. Presented in part at the 26th Annual Meeting of the Tissue Culture Association, at Montreal, Canada, 2–5 June 1975. This investigation was supported by U.S. Public Health Service Research Grant No. AI 09914 from the National Institute of Allergy and Infectious Diseases. This is Paper No. 9416, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells

Summary The ability of selected vasoactive agents to influence cyclic AMP levels of confluent, early-passaged bovine calf aortic and pulmonary artery endothelial cells was investigated. Among the agents tested, only the catecholamines (isoproterenol, epinephrine, nonrepinephrine) and prostaglandins (PGE₁, PGE₂, PGF_2a) resulted consistently in increased cyclic AMP production in both cell populations. The degree of cyclic AMP stimulation obtained with other vasoactive compounds (angiotensins I and II, bradykinin, and serotonin) tended to be either very small or difficult to reproduce. Isoproterenol stimulation was blocked completely by propanolol, a β-blocking agent, but not by phentolamine, an α-blocking agent. These results reveal that bovine calf aortic and pulmonary artery endothelial cells are responsive to catecholamines and prostaglandins, and therefore presumably possess both sensitive adenylate cyclases and plasma membrane receptors for these compounds. This work was supported by a Young Investigator Grant HL-21189 from the National Institutes of Health, United States Public Health Service.     

A method for the continuous cultivation of mammary epithelium

Summary Established cell lines of mammary epithelium have been obtained from mice, rats, and hamsters. Maintenance and replication of the epithelium in serial subcultures were dependent on their periodic treatment with collagenase. Because collagenase is not cytotoxic and has maximum efficiency at neutral pH in isotonic saline solutions containing calcium and magnesium, this enzyme can be introduced directly into the culture medium; cells have been maintained for 3 days in such a medium with serous enrichment at no detriment to them. Up to 10% concentrations of serum have not interfered with enzymatic activity. Supported by United States Public Health Service Grant CA-08515 and CA-08740 from the National Cancer Institute. General Research Support Grant FR-5582 from the National Institutes of Health, and Grant-in-Aid Contract M-43 from the State of New Jersey.     

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye,ruby-eye-2-J,and maroon

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. Concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney -glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are now known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.This work was supported in part by United States Public Health Service Research Grant GM-19521 and by National Science Foundation Grant PCM77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM07093-03. F. W. was a high school student in the summer program supported by National Science Foundation Grant SP177-26980.     

Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERα and ERβ by immunohistochemistry and in situ hybridization. ERβ, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERβ-positive cells increased significantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17β-estradiol (E₂), but not E₂ + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a significant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .