Showing papers in "Endocrinology in 1986"

Purification, Characterization, and in vitro Differentiation of Cytotrophoblasts from Human Term Placentae*

Abstract: Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.

Glucagon-Like Peptides GLP-1 and GLP-2, Predicted Products of the Glucagon Gene, Are Secreted Separately from Pig Small Intestine but Not Pancreas

Abstract: We developed specific antibodies and RIAs for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), two predicted products of the glucagon gene, and studied the occurrence, nature, and secretion of immunoreactive GLP-1 and GLP-2 in pig pancreas and small intestine Immunoreactive GLP-1 and GLP-2 were identified in glucagon-producing cells of the pancreatic islets, and in glicentin-producing cells of the small intestine Immunoreactive GLP-1 and 2 in intestinal extracts corresponded in molecular size to peptides synthesized according to the predicted structure By reverse phase HPLC, intestinal and synthetic GLP-1 behaved similarly, whereas synthetic and intestinal GLP-2 differed Pancreatic extracts contained a large peptide with both GLP-1 and GLP-2 immunoreactivity Secretion was studied using isolated perfused pig pancreas during arginine stimulation, and isolated perfused pig ileum during either luminal glucose stimulation or vascular administration of the neuropeptide, gastrin-releasing peptide (GRP) Im

Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone.

Abstract: Indirect evidence suggests that cells of the osteoblastic lineage may mediate augmented osteoclastic bone resorption induced by PTH. To test this suggestion, osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Resorption was measured by computer-assisted morphometric and stereophotogrammetric quantification of osteoclastic excavations, identified in the scanning electron microscope after culture. We compared the effect of PTH on bone resorption by osteoclasts incubated alone with the effect of the hormone on resorption by osteoclasts cocultured with osteoblastic cells. PTH had no effect on bone resorption by osteoclasts alone, but in the presence of any of three osteoblast-containing cell populations, or in the presence of cloned, hormone-responsive osteosarcoma cells, PTH caused a 2- to 4-fold increase in osteoclastic resorption. Significant stimulation was observed at 10−4 IU/ml PTH. None of the osteoblastic cell populations caused morpholo...

Effect of pH on Bone Resorption by Rat Osteoclasts in Vitro

Abstract: Acidosis has been implicated in the pathogenesis of various metabolic bone diseases. Experimental acid ingestion is known to stimulate the release of bone mineral in humans and increase osteoclastic resorption surfaces in animals, but the mechanism has remained unclear. We have investigated the effect of medium pH on osteoclastic activity in vitro using a new method that allows direct measurement of bone resorption. Osteoclasts were disaggregated from neonatal rat long bones and allowed to settle onto thin slices of bovine cortical bone. After 24-h incubation in medium 199, adjusted to the required pH, bone slices were fixed, and the number and surface area of resorption pits per slice were determined. Reduction in medium pH from 7.4 to 6.8 resulted in a 14-fold increase in the mean area resorbed per bone slice (P less than 0.01). Five- and 9-fold increases were seen at pH 7.2 and 7.0, respectively. Incubation of bone cells with human PTH-(1-34) (100 ng/ml) did not significantly enhance resorption at either pH 7.4 or 6.4, whereas human calcitonin (50 pg/ml) produced near-total inhibition. Mean osteoclast and mononuclear cell numbers were unaltered by all treatments. This is the first direct demonstration that low pH stimulates cell-mediated bone resorption, an observation that may have considerable physiological and pathophysiological significance.

Ontogeny of the stress response in the rat: role of the pituitary and the hypothalamus.

Abstract: The neonatal rat shows a period of decreased responsiveness to noxious stimuli during the first 3 weeks of life, but the nature of this impairment is still controversial. To test the functionality of the hypothalamus-pituitary-adrenal axis during this period, we studied pituitary and adrenal responsiveness to exogenous ovine CRF and the ability of various stressors (ether vapors, electroshocks, and hypoxia) to elicit ACTH and corticosterone secretion. We also measured hypothalamic CRF content and pituitary ACTH content as well as CRF-binding sites in the anterior pituitary. From days 3–10, small elevations in plasma ACTH and corticosterone levels were observed after a 3-min exposure to ether vapors or electroshocks. In contrast, during this period, a 20- min exposure to hypoxia (5% O2 in N2) was unable to trigger measurable ACTH secretion, while corticosterone was significantly elevated. From days 14–21, plasma ACTH and corticosterone levels increased significantly after exposure to ether stress, hypoxia,...

1,25-Dihydroxyvitamin D3 and human bone-derived cells in vitro: effects on alkaline phosphatase, type I collagen and proliferation.

Abstract: 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], but not 24,25-(OH)2D3, stimulates the alkaline phosphatase activity of cultured human bone cell populations. The stimulatory effect of the sterol was dose dependent (10−10–10−7 m), evident by 24 h, and observed over a range of cell densities. Analysis of the radiolabeled collagens synthesised by human bone cell cultures indicated the synthesis of predominantly type I collagen. In the presence of l,25-(OH)2b3, but not 24,25-(OH)2D3, there was a dose-dependent (10−11–10−9 m) increase in radiolabeled proline incorporation into collagenase-digestible protein and in the amount of collagen synthesized, expressed as a percentage of the total protein synthesis. The effect of 1,25-(OH)2D3 was observed over a range of cell densities and appeared to be specific for the synthesis of type I collagen. The stimulatory effect of 1,25- (QH)2D3 on alkaline phosphatase activity and the increase in proline incorporation into collagenase-digestible protein were accompanied by a dose-de...

Induction of Epidermal Growth Factor-Related Polypeptides by 17β-Estradiol in MCF-7 Human Breast Cancer Cells

Abstract: MCF-7 human breast cancer cells release polypeptides related to insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with 17β-estradiol, which is required in vivo for MCF-7 tumor growth in the nude mouse and stimulates the MCF-7 growth rate in vitro, resulted in selectively enhanced growth factor activities in CM. Autostimulatory growth-promoting activity was elevated at least 2-fold, and EGFlike polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor- reactive EGF-like polypeptides, suggestive of high molecular weight transforming growth factorα, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and fibroblast-transforming activity. It is possible that estradiol stimulation of MCF- 7 growth and/or tumor formation may depend on...

Osteoblast-Like Cells in the Presence of Parathyroid Hormone Release Soluble Factor that Stimulates Osteoclastic Bone Resorption

Abstract: PTH stimulates osteoclastic bone resorption in viuo and in organ culture. We have previously found that if osteoclasts are disaggregated from bone and incubated on bone slices, PTH does not increase bone resorption, but does so if osteoblastic cells are added to the cultures. This suggests that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption. In the present paper we describe investigations into the mechanism by which osteoblastic cells stimulate osteoclasts. We found that increased resorption could not be accounted for by changes in the bone substrate. Osteoblast-like cells (UMR106) incubated with PTH did, however, release a factor into the culture supernatant that stimulated osteoclastic bone resorption. This factor was stable for at least 7 days when stored at 4 C and survived freeze-thawing, but was inactivated by heating to 65 C for 30 min. Activity was lost entirely after dialysis using a Spectrapor membrane with a mol wt c...

Autocrine Growth Stimulation of the MCF 7 Breast Cancer Cells by the Estrogen-Regulated 52 K Protein

Abstract: The growth of MCF 7 human breast cancer cells is stimulated in vitro by estradiol (E2) and we have previously shown that estrogen-regulated glycoproteins released into the culture medium can partly mimick this effect. In this paper, we evaluate the mitogenic activity of the 52 K glycoprotein, which is a major E2-stimulated protein released by MCF 7 cells. The 52 K protein was purified 600-fold by affinity chromatography on Concahavalin A and an anti-52 K monoclonal antibody Sepharose columns. The 99% purified 52 K protein fraction stimulated the growth of estrogen-deprived MCF 7cells. A mean 1.7-fold increase was obtained with nanomolar concentrations of seven different preparations of 52 K protein. This stimulation represented 40% of the mitogenic effect of E2. Both the 52 K protein and E2 induced microvilli at the cell surface but the effect of the 52 K protein ocurred earlier. Other putative growth factors which are also stimulated by E2 and observed by [35SJcysteine labeling did not comigrate with the...

Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves'-IgG.

Abstract: The present studies were undertaken to examine the factors that influence the growth of cells of endocrine gland origin, particularly the possible interactions between "nonspecific" growth factors and the trophic hormone for a target endocrine cell. As a model system, we explored the individual and conjoint effects of insulin-like growth factor-I (IGF-I) and TSH on the growth of FRTL5 cells, a nontransformed line of cloned rat thyroid follicular epithelium. In these cells, IGF-I and TSH each produced a dose-dependent enhancement of DNA synthesis and cell proliferation. When added together, IGF-I and TSH were markedly synergistic in stimulating DNA synthesis, producing increases in 3H-thymidine incorporation that were far greater than the sum of the effects of each alone. A similar effect of IGF-I was evident in the case of the stimulation of DNA synthesis produced by immunoglobulin G (IgG) preparations from the blood of patients with Graves' disease. Such IgG bind to the TSH receptor and mimic the actions of TSH therein. It is suggested, therefore, that there exist in the FRTL5 cell line at least two mechanisms for the regulation of growth, one activated at the level of the IGF-I receptor and the other at the level of the TSH receptor. When the two pathways are activated concurrently, a synergistic enhancement of DNA synthesis takes place. The findings indicate that the FRTL5 cell line is an excellent model in which to study these complex interactions and that IGF-I may be a determinant of thyroid cell growth, both normally and in certain thyroid diseases.

Vitamin D3 improves impaired glucose tolerance and insulin secretion in the vitamin D-deficient rat in vivo

Abstract: It has previously been shown in this laboratory that vitamin D3 is essential for normal insulin secretion from the perfused rat pancreas. In this present study, the influence of vitamin D status on insulin secretion in vivo was investigated. Intravenous glucose tolerance tests were performed on conscious vitamin D-deficient rats (-D), vitamin D-replete rats fed ad libitum (+D AL), and vitamin D-replete rats pair fed to the D-deficient animals (+D PF). Vitamin D deficiency, easily recognizable by low daily dietary intake and depressed plasma calcium levels, was found to impair plasma glucose clearance as characterized by an elevated KG value (representing a function of the area beneath the tolerance curve). KG values for the +D AL, +D PF, and -D groups were 504 +/- 15, 480 +/- 46, and 641 +/- 28, respectively. The increase in KG corresponded to a significant reduction in glucose-mediated insulin secretion as compared to the +D animals. This difference appeared not to be related to the increase caloric intake associated with vitamin D repletion, since +D rats which had been pair fed to the -D animals also exhibited restored plasma insulin levels in response to glucose. Plasma phosphorus concentrations were comparable in all three groups, and thus this parameter is also unlikely to be a contributory factor in the observed phenomenon. Additional experiments were conducted to evaluate the involvement of hypocalcemia in the observed impaired glucose tolerance. Normalization of plasma calcium levels (from 4.8 mg/100 ml to 9.6/100 ml) of the -D rats, by dietary calcium and phosphorus manipulation, failed to improve glucose clearance (KG for -D normocalcemic rats = 639 +/- 61) or insulin secretion. These results support the concept that vitamin D plays a physiological role in insulin secretion, acting, at least in part, independently of nutritional factors and the prevailing plasma calcium and phosphorus concentrations.

Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels.

Abstract: Somatomedin preparations have previously been shown to suppress GH release. The effects of a synthetic recombinant human insulin-like growth factor analog (IGF-I; Thr 59) were, therefore, tested on long term GH secretion by male rat pituitary monolayer cell cultures grown in serum-free defined medium. IGF-I (3.25 nm) maximally suppressed basal GH secretion for up to 72 h by 66%, with an ED50 of 0.1 nm. Human pancreatic GRF-(l–44) (GHRH; 1 nm) stimulated GH secretion by 230% during 72 h. IGF-I (0.13 nm) suppressed GHRHstimulated GH secretion by 30% (P < 0.005). IGF-I (0.625 nm) completely abolished stimulation of GH by GHRH (1 nm), while higher doses of IGF-I (up to 6.5 nm) actually suppressed GH secretion even in the presence of GHRH (up to 1 nm). The depletion of intracellular GH levels in GHRH-treated cells was reversed by IGF-I (3.25 nm). PRL secretion was not altered in the same cells by IGF-I. Equivalent doses of epidermal growth factor and fibroblast growth factor did not alter basal or GHRHstimulat...

Suppression by 1,25(OH)2D3 of transcription of the pre-proparathyroid hormone gene.

Abstract: When bovine parathyroid cells in culture are exposed to the active vitamin D metabolite 1,25(OH)2D3, a significant decrease in the steady–state levels of pre–proparathyroid hormone (pre–proPTH) mRNA occurs. The possibility that the fall in specific mRNA is due to a decrease in rate of transcription of the PTH gene was examined in this study. In the presence of 1,25(OH)2D3, there was a rapid and steady decline in PTH gene transcription rate which fell to a minimum of 10–15% of control at 24 h. The effect was observed at physiological levels (10−11m) and was also fully reversible.

Growth hormone increases ovarian levels of immonoreactive somatomedin c/insulin-like growth factor i in vivo.

Abstract: The hypothesis that GH may affect gonadal function by increasing local levels of the GH-dependent somatomedin C/insulin-like growth factor I (IGF I) was tested. Ovine GH (200 μg) was injected into immature, hypophysectomized, estrogen-treated female rats; animals were sacrificed 8 or 12 h later. Renal and ovarian homogenates were acid extracted and chromatographed over Sephadex G-50. Eight h after GH injection, 3.6 to 6.4-fold increases in immunoreactive IGF I (IR-IGF I) levels were observed in either ovarian or renal extracts subjected to acid chromatography. Twelve h after GH treatment, IR-IGF I levels remained elevated, but were lower than after 8 h. In neither case could IR-IGF I levels be accounted for by serum contamination. IR-IGF I eluted with an apparent mol wt near that of synthetic human IGF I in both kidney and ovary. Thus, GH can directly increase ovarian and renal tissue IR-IGF I levels in vivo. Taken with previous observations showing a direct gonadotropin-enhancing effect of IGF I on rat g...

Placental protein 12 is a decidual protein that binds somatomedin and has an identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid.

Abstract: Placental protein 12 (PP12) was originally isolated from term human placenta and adjacent membranes. Recently we found that the site of PP12 synthesis is decidua but not placenta. In this work, the purity of PP12 was first tested by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis and by reverse phase HPLC, and the N-terminal amino acid sequence of 15 residues was determined by a liquid-phase sequencer. A single amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala-Asp-Glu-Leu-Ala-Leu was obtained showing identity to the known N-terminal amino acid sequence of somatomedin-binding protein from human amniotic fluid. Like the latter, PP12 bound somatomedin (insulin-like growth factor I) as demonstrated in gel chromatography by a shift in the elution pattern of [125I]iodo-insulin-like growth factor I after incubation with PP12. These data show that PP12 is a somatomedin-binding protein and extend through previous literature on PP12 the existing knowledge on the physiology and pathophysiology of somatomedin-binding protein(s) in human reproduction and cancer.

Rapid in Vivo Effects of Glucocorticoids on the Integrity of Rat Lymphocyte Genomic Deoxyribonucleic Acid

Abstract: Glucocorticoid-induced lymphocytolysis has been studied for many years; however, the mechanism of lymphoid cell death has not been elucidated. In this study we have investigated the effects of glucocorticoids on the lymphocyte genome using the rat thymocyte model. Adrenalectomized rats were injected ip with dexamethasone (DEX) and killed thereafter. The thymus gland was removed, and DNA was extracted from isolated thymocytes and then separated electrophoretically on 1.8% agarose gels. Administration of glucocorticoids in vivo resulted in the cleavage of lymphocyte DNA at internucleosomal intervals. Genomic DNA separated on agarose gels from DEX-treated rats appeared as a ladder of DNA fragments which were multiples of about 180 base pairs, while DNA from control rats appeared as a single high mol wt band. This cleavage of thymocyte DNA was a rapid effect of adrenal steroid treatment and occurred before cell death. Thymocyte DNA fragmentation increased with time after DEX treatment and the dose of half-maximal response in vivo was estimated to be about 1.8 X 10(-8) M. Internucleosomal cleavage of DNA was only observed in lymphoid tissues (thymus and spleen), but not in other glucocorticoid-sensitive tissues (kidney, liver, heart, brain, or testis). Treatment of rats with estrogen, androgen, or progestin failed to elicit thymocyte DNA degradation. Glucocorticoid-induced DNA cleavage was partly inhibited by the glucocorticoid antagonist RU 486 (17 beta-hydroxy-11 beta,4-dimethylaminophenyl-17 alpha-propynl-estra-4,9-diene-3-one). These findings suggest that glucocorticoids activate, via a receptor-mediated process, an endonuclease-like activity in lymphoid tissues which cleaves the lymphocyte genome at internucleosomal sites. Activation of this nuclease by glucocorticoids precedes lymphocytolysis and may represent the hormonal regulation of programmed cell death.

The temporal response of bone to unloading

Abstract: Rats were suspended by their tails with the forelimbs bearing the weight load to simulate the weightlessness of space flight. Growth in bone mass ceased by 1 week in the hindlimbs and lumbar vertebrae in growing rats, while growth in the forelimbs and cervical vertebrae remained unaffected. The effects of selective skeletal unloading on bone formation during 2 weeks of suspension was investigated using radio iostope incorporation (with Ca-45 and H-3 proline) and histomorphometry (with tetracycline labeling). The results of these studies were confirmed by histomorphometric measurements of bone formation using triple tetracycline labeling. This model of simulated weightlessness results in an initial inhibition of bone formation in the unloaded bones. This temporary cessation of bone formation is followed in the accretion of bone mass, which then resumes at a normal rate by 14 days, despite continued skeletal unloading. This cycle of inhibition and resumption of bone formation has profound implication for understanding bone dynamics durng space flight, immobilization, or bed rest and offers an opportunity to study the hormonal and mechanical factors that regulate bone formation.

A Possible Mechanism for Inhibition of Angiogenesis by Angiostatic Steroids: Induction of Capillary Basement Membrane Dissolution*

Abstract: A new class of angiostatic steroids acts independently of previously identified steroid functions to inhibit angiogenesis when administered with heparin. Development of angiostatic steroids as therapeutic modulators of blood vessel growth would be greatly facilitated if their mode of action were thoroughly understood. However, the mechanism by which these steroids produce capillary regression is not known. The distributions of fibronectin and laminin were studied in growing and regressing capillaries by immunofluorescence microscopy to determine whether capillary basement membrane (BM) alterations could be involved in the mechanism of antiangiogenesis. In normal 8-day-old chick chorioallantoic membrane, fibronectin and laminin appeared in continuous linear patterns within BM surrounding growing capillaries. In contrast, chorioallantoic membranes treated with combinations of angiostatic steroid and heparin exhibited capillary BM fragmentation and eventually complete loss of fibronectin and laminin from reg...

Specific Receptor and Cardiovascular Effects of Calcitonin Gene-Related Peptide

Abstract: Specific binding sites for calcitonin gene-related peptide (CGRP) were demonstated in the rat heart and spleen. Autoradiography revealed rat [125I]iodo CGRP binding associated with the intima and media of the aorta, the coronary arteries and the heart valves, and the red pulp of the spleen. Halfmaximal inhibition of rat [125I]iodo-CGRP binding to membranes of the rat atria and the spleen was obtained with, respectively, 5 and 0.35 nm unlabeled rat CGRP; these values correspond to ECso values of 3 and 0.14 nm for activation of adenylate cyclase by CGRP. In the isolated, spontaneously beating right atrium, the EC50 values of stimulation of the force and rate of contraction by rat CGRP were 120 and 70 nm, respectively. Rat CGRP caused relaxation of splenic strips, precontracted with noradrenaline; the ECso was 50 nm.ECso The β-adrenergic blocking agent metoprolol, while obliterating the increase in the force and rate of contraction evoked by noradrenaline in the right atrium, did not significantly change the...

Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture: a comparative study.

Abstract: Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were evaluated by using purified adult Leydig cells from Sprague-Dawley rats. Purified Leydig cells were found to contain both high and low affinity binding sites for insulin, with Ka values of 1.08 X 10(9) and 1.1 X 10(7) M-1, respectively. Using affinity cross-linking of [125I]iodoinsulin to plasma membrane insulin receptor, several bands were identified by autoradiography under nonreduced conditions with mol wt of 230,000, 280,000, and 300,000. After reduction with 50 mM dithiothreitol, only one band was identified with a mol wt of 130,000, consistent with the alpha-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 X 10(9) M-1. Multiple high mol wt bands (greater than 250,000) were identified under nonreduced conditions by affinity cross-linking. Under reduced conditions, one band with an approximate mol wt of 135,000 was identified. Purified Leydig cells (10(5) cells/ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 Nutrient Mixture (1:1) containing 0.1% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2-95% air. Insulin and IGF-I stimulated testosterone formation as early as 3 h after administration, and their effects were completely blocked by the addition of a protein synthesis inhibitor, cycloheximide (1 microgram/ml). Insulin and IGF-I also significantly potentiated hCG-and 8-bromo-cAMP-induced testosterone formation. Furthermore, insulin and IGF-I potentiated hCG-stimulated cAMP formation. This suggests that insulin and IGF-I have effects at both the LH receptor sites and the steps beyond adenylate cyclase. The ED50 values of insulin and IGF-I-stimulated testosterone formation were comparable (25 ng/ml). In conclusion, we found that Leydig cells contain specific insulin and type I IGF receptors, and both insulin and IGF-I are capable of modulating Leydig cell steroidogenesis.

Evidence for a Physiological Role for Oxytocin in the Control of Prolactin Secretion

Abstract: The presence of oxytocin (OT) in neuronal elements of the external layer of the median eminence and in hypophysial portal plasma suggests a role for the peptide in the control of anterior pituitary function. We have reported previously that OT stimulates PRL release in vitro; therefore, we attempted to establish evidence for a physiological PRL-releasing role for OT. Plasma OT levels rose significantly just before the PRL surges occurring during a suckling stimulus in lactating rats (10 min after pup reinstatement vs. 15 min for PRL) and 48 h after estrogen injection in ovariectomized (OVX) rats (at 1200 h vs. 1300 h). Dispersed anterior pituitary cells harvested from lactating female rats and OVX estrogen-primed rats released PRL in a specific, significant, and dose-related fashion when perifused in vitro with incubation medium containing 10−7–10−9 m OT, doses similar to levels found previously in hypophysial portal plasma. Infusion of antiserum specific for OT into lactating females before pup reinstate...

The effect of iodide ingestion on the development of spontaneous lymphocytic thyroiditis in the diabetes-prone BB/W rat.

Abstract: It has been suggested that the incidence of Hashimoto's thyroiditis is increased in the presence of high iodide intake. The diabetes-prone BB/W rat develops spontaneous histological autoimmune lymphocytic thyroiditis (LT) without functional hypothyroidism between 60 and 120 days of age. Studies were carried out to determine whether iodine administration to BB/W rats would affect the incidence and severity of LT and induce hypothyroidism. Iodide (0.05% in water) or tap water (C) was administered ad libitum to 42 10-month-old BB/W rats and 71 30-day-old BB/W rats for 8 weeks. For control purposes, 0.05% iodide or tap water (C) was also administered ad libitum to 42 30-day-old nondiabetic and non-LT-prone BB/W genetically equivalent rats (W-line) for 12 weeks and 41 21-day-old Wistar rats for 7 weeks. In a separate experiment, weanling BB/W rats were fed a low iodine diet, a control iodine-sufficient (C) diet, or Purina chow (P) and tap water ad libitum for 8 weeks. In each experiment, blood was obtained at the time of death for the measurement of serum T4, T3, TSH, and antithyroglobulin antibody (anti-Tg Ab), and the thyroids were removed for histological evaluation (0 = no LT; 1-4 = LT). Iodide administration (0.05%) induced a significant increase in the incidence of LT in 30-day-old BB/W rats (I, 77%; C, 30%, P less than .001). Thyroid weight and serum T4, T3, and anti-Tg Ab concentrations were not affected by iodide administration. However, the presence of LT was associated with a significant increase in thyroid weight and anti-Tg Ab concentrations. BB/W rats subjected to a low iodine diet exhibited a significantly decreased incidence of LT (low I, 8.6%; C, 47.3%; P less than 0.01), but no statistically significant difference in anti-Tg Ab levels. Increased iodide intake did not significantly affect the incidence of LT in adult BB/W rats and did not induce LT or affect thyroid function in W-line or Wistar rats. These data show that iodine intake significantly affects the incidence of spontaneous LT in young, genetically predisposed rats.

Hormonal Regulation of Granulosa Cell Inhibin Biosynthesis

Abstract: The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Food-Restricted, Prepubertal, Female Rats: Rapid Recovery of Luteinizing Hormone Pulsing with Excess Food, and Full Recovery of Pubertal Development with Gonadotropin-Releasing Hormone*

Abstract: Prepubertal female rats were maintained continuously at 45% of their expected 50-day body weight by restricting their food intake. Uteri and ovaries declined in weight under these conditions. No evidence of pulsatile LH release was seen when these animals were examined at 50 days of age. Allowing unlimited access to food at this time caused rapid pubertal development. LH pulsing began in some females within 12 h; strong LH pulsing was seen in most females within 24 h, and all ovulated after only 2 1/2 or 3 1/2 days of ad libitum feeding. These were fertile ovulations, accompanied by mating and resulting in pregnancy. Administering GnRH in a pulsatile manner to 50-day-old, food-restricted animals also yielded full pubertal development. Uteri and ovaries gradually increased in weight, and ovulation occurred in 3 1/2 to 5 1/2 days. These findings support a contention that the major reproductive deficit resulting from food restriction relates to the control of GnRH secretion. In toto they also suggest a close metabolic coupling between some dimension of nutrient and/or energy processing and the GnRH pulse generator in the normally growing female.

Human Platelet-Derived Transforming Growth Factor-βStimulates Parameters of Bone Growth in Fetal Rat Calvariae

Abstract: Human platelet-derived transforming growth factor type β (TGFβ) is mitogenic for fetal rat calvariae in serum-free organ culture. It enhances DNA synthesis in short (24-h) and long (48- to 96-h) term cultures, but produces no significant stimulatory effects on bone collagen synthesis or alkaline phosphatase activity (two parameters of differentiated osteoblastic cell-type function) when present continuously in culture. Transitory treatment with TGFβ, however, induces a subsequent stimulation of collagen and noncollagen protein synthesis that depends on prior cell replication, suggesting an increase in the number of newly differentiated bone cells. In addition, TGFβ increases prostaglandin release, but this effect is probably unrelated to its mitogenic function. TGFβ activity is also found in culture medium conditioned by fetal rat calvariae, and the bone-derived factor produces effects similar to those of the human platelet factor. This polypeptide, therefore, may have an important function in early stage...

Secretory endometrium synthesizes placental protein 14.

Abstract: Saline extracts of human nonpregnant endometrium were found to contain placental protein 14 (PP14). The tissue PP14 content was highest in the late secretory phase (median, 7.7 mg/g protein; n = 14), whereas proliferative endometrium (n = 8) was either PP14 negative or showed a low PP14 content (median, 0.15 mg/g protein). By immunoperoxidase staining, PP14 was localized in the glandular epithelial cells of endometrium. In tissue culture, secretory endometrium released more PP14 than proliferative endometrium, and cycloheximide markedly decreased this release. Synthesis of PP14 by secretory endometrium was demonstrated by incorporation of [35S]methionine into immunoprecipitable PP14. These results show that PP14 is synthesized and secreted by the nonpregnant endometrium. (Endocrinology 118: 1782–1786, 1986)

Intracerebroventricular Infusion of Aldosterone Induces Hypertension in Rats

Abstract: There is suggestive evidence for the direct participation of mineralocorticoids in the production of centrally mediated hypertension. Unilaterally nephrectomized Sprague-Dawley rats received a continuous infusion for 30 days using implanted osmotic minipumps with 1) artificial spinal fluid (CSF) intracerebroventricularly (icvt); 2) 0.005 μg aldosterone/h icvt; 3) 0.005 μg aldosterone/h sc; 4) 0.5 μg aldosterone/h sc. There was no significant difference between the groups in average weight gain (52 ± 2 g) or organ weight to body weight, nor was urine volume increased above normal except in the group receiving the high sc dose of aldosterone. Blood pressure was significantly elevated only in those animals receiving 0.005 /g aldosterone/h icvt and 0.5 /g aldosterone/h sc. A second experiment was done using a specific spironolactone-type mineralocorticoid antagonist, prorenone. The rats were grouped as follows: 1) CSF icvt; 2) 0.005 μg/h aldosterone icvt; 3) 0.005 μg/h aldosterone plus 0.005 μg/h prorenone ic...

Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

Abstract: Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha-hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development.

Preparation of monoclonal antibodies to the avian progesterone receptor.

Abstract: In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated αPR 6, 11, 13,16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradien...

Interleukin-1 has independent effects on deoxyribonucleic acid and collagen synthesis in cultures of rat calvariae.

Abstract: Interleukin-1 (IL-1), a monokine known to be important in host defense mechanisms and recently reported to stimulate bone resorption, was studied for its effects on bone formation in cultures of 21-day-old fetal rat calvariae. IL-1 at 0.1–5 U/ml stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA) by 29–123% in calvariae treated for 24–96 h. IL-1 also increased the bone DNA content and the number of mitoses after colcemid arrest. IL-1 stimulated total protein synthesis. Treatment with IL-1 at 0.01–1 U/ml for 24 h caused a small increase in the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). However, higher doses of IL-1 (5 U/ml) or longer exposure to the agent (1 U/ml for 96 h) inhibited the labeling of CDP but not of NCP. IL-1 affected only type I collagen. The stimulatory effects of IL-1 on DNA, CDP, and NCP labeling were independent, since they were observed at different doses, and hydroxyurea abolished the effect on DNA...