Extracts Of Pituitary Glands Research Articles

Induced reproduction in Brycon cf. erythropterus

Doses of pituitary gland extracts from 3.5 to 10 mg per kg fresh weight are reported to induce spawning in a Brycon species. When breeders were held in ponds and were fed with dry diet, good results were obtained with regard to egg quality and fertilization, and hatching rates were up to 80%. Incubation of eggs, embryonic development and rearing of larvae and juveniles on zooplankton and a dry feedstuff are described. A method is proposed to separate mature from immature specimens prior to the hormone application.

A hypothalamic-pituitary system that stimulates the release of plasminogen activator in the rat

We have studied the possible role of the hypothalamic-pituitary system in the control of the release of plasminogen activator (PA) into peripheral blood of male rats. Plasminogen activator was measured by euglobulin lysis time. Desamino- d-arginine 8 vasopressin (dDAVP) and adrenaline injected i.v. induced an increase in plasma PA as did electrical stimulation of the median eminence (ME), but dDAVP had no effect on plasma PA in hypophysectomized rats. The PA response to ME stimulation was similar in Brattleboro rats (deficient in vasopressin) and adrenalectomized Wistar rats compared with intact Wistar rats, but was abolished by section of the pituitary stalk and was negligible in hypophysectomized rats. The 41-residue corticotropin releasing factor (CRF) had no effect on PA release. Saline extracts of anterior pituitary gland from both normal Wistar and Brattleboro rats produced a dose-dependent increase in plasma PA when injected into normal Wistar rats. The activity of pituitary tissue was abolished by boiling, but not by di-isopropyl fluorophosphate which inactivates PA itself. Thus the anterior pituitary gland of the rat contains a heat-labile factor which stimulates the release of PA from peripheral stores into the circulation. This pituitary factor is released by a hypothalamic factor that is neither vasopressin nor CRF.

Oxytocin content of human fetal pituitary glands

Seventeen human fetal and neonatal pituitary glands were removed at necropsy, and analyzed for oxytocin content by a specific and sensitive radioimmunoassay, after homogenization in 0.4M acetic acid. Serial dilution of the pituitary extract showed parallelism with the oxytocin standard curve on the radioimmunoassay. Column chromatography of the pituitary gland extract gave a single peak of immunoreactive oxytocin as determined by the radioimmunoassay. In eight fetuses, 14 to 17 weeks' gestation, pituitary gland oxytocin was 10.2 ± 5.9 ng/gland (mean ± SE), whereas an 18-week fetus had 5.8 ng of oxytocin per gland. Pituitary gland oxytocin content increased to 38.4 ng/gland in a 20-week fetus removed at hysterectomy, and 31.6 ng/gland in a 26-week fetus. Fetal pituitary gland oxytocin values were 22.1 ng/gland and 57.0 ng/gland at 32 weeks and increased significantly to 544.3 ± 33.8 ng/gland in 1- to 5-day-old term newborn infants (n = 3). However, a 13-day-old term newborn infant had 3.7 ng of oxytocin per pituitary gland. The increased pituitary gland oxytocin content with advancing gestation was due to a significant increase in oxytocin concentration rather than to an increase in the weight of the pituitary gland. The findings indicate that oxytocin is present in fetal pituitary glands as early as 14 to 17 weeks' gestation and increases at term to 50 and 13 to 14 times more than in early midtrimester and early third-trimester pregnancy, respectively.

Identification of trypsin inhibitor in bovine pituitary extracts as a survival factor for adult rat hepatocytes in primary culture.

Adult rat hepatocytes in primary culture cannot survive more than 2 days in the absence of calf serum. An extract of bovine pituitary gland had a similar effect to calf serum on the survival of hepatocytes, but its specific activity was 70 times that of calf serum. The survival factor was purified from bovine pituitary gland, and obtained in a homogeneous state judging by sodium dodecylsulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography. Its purification was achieved by acid extraction of the pituitary gland, gel filtrations on Sephadex G-75 and Ultrogel AcA 202, ion-exchanger chromatography on CM-cellulose, and then reverse phase high performance liquid chromatography. The purified factor was effective at 10 ng/ml and maximally effective at 100 ng/ml. The overall recovery of its activity was 30%, and the specific activity of the purified factor was 3,100 times that in the acid extract. The molecular weight (Mr 8,000), estimated by Sephadex G-200 filtration, and the strong basic character (pI 10.5) of the purified survival factor suggested that it may be the trypsin inhibitor found in various tissues. Indeed, the amino acid composition of the pure material was identical with that of bovine pancreatic trypsin inhibitor (bPTI). The purified survival factor had the same inhibitory activity on trypsin as commercial bPTI and, conversely, commercial bPTI greatly enhanced survival of rat hepatocytes. Thus, the survival factor in bovine pituitary gland was identified as bPTI.

Tissue distribution and molecular forms of a novel pituitary protein in the rat.

A sensitive and specific radioimmunoassay (RIA) was developed for a novel pituitary protein that we recently isolated from human and porcine pituitary gland and designated 7B2. By employing this RIA, we were able to detect and assay this novel protein in different rat tissue extracts. The concentrations of 7B2 in rat anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland were 10,400 +/- 804; 6,190 +/- 908; 773 +/- 50; 697 +/- 83 and 1,368 +/- 116 pg/mg tissue (wet weight, n = 10, mean +/- SEM), respectively. However, the concentrations of 7B2 were lower than 30 pg/mg tissue in extracts of pancreas, ileum and colon, and were below the sensitivity of the RIA in extracts of liver, kidney, spleen, lung, adrenal cortex and testis. Gel permeation chromatography of extracts of anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland on Sephadex G-100 revealed that most of the immunoreactive (Ir)-7B2 has an apparent molecular weight of 45,000-50,000. Subsequent dissociation of this Ir-7B2 by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS) yielded an Ir-7B2 with an apparent molecular weight of around 19,000. In addition, high K+ concentration (50 mM) induced the release of Ir-7B2 from cultured cells of both rat anterior pituitary and neurointermediate lobe. Finally, Ir-7B2 was detected in the neurosecretory granule fraction prepared from porcine neurointermediate lobe. These results indicate that 7B2 may be a novel secretory protein in the pituitary gland.

The hypogonadal mouse pituitary contains bioactive LH.

Saline extracts of pituitary glands from normal and hpg male mice were assayed for LH by the testosterone production assay. The extract of hpg pituitary tissue stimulated testosterone production by mouse Leydig cells in a dose-dependent manner which paralleled that of normal mouse pituitary. The normal mouse pituitary was about 20 times as potent as hpg pituitary which is similar to the relative potency obtained by radioimmunoassay. These results show that differentiation of the gonadotrophs and the synthesis of some biologically active LH can occur in the absence of LHRH.

Study of prolactin levels in the ferret.

A radioimmunoassay for canine prolactin has been used to measure prolactin in the ferret. Serial dilutions of extracts of ferret pituitary glands and of ferret plasma yielded curves that were parallel with the canine prolactin standard curve. The sensitivity, accuracy, reproducibility and precision of the assay were within acceptable limits. Plasma prolactin levels increased after the administration of thyrotrophin releasing hormone (TRH) or chlorpromazine, but not after giving luteinizing hormone releasing hormone. Female ferrets, which were anoestrous, oestrous or spayed, and male ferrets had similar basal prolactin levels when sampled under sodium pentobarbitone anaesthesia. These basal levels were higher than in conscious males and the latter also showed a lesser response to TRH. Hypophysectomy significantly reduced basal prolactin levels in female ferrets by 2 h postoperatively and abolished the response to TRH.

Studies of calcium-binding protein in cattle.

We have developed and validated a double-antibody radioimmunoassay for quantifying bovine calcium-binding protein (CaBP). Cross-reactivity between the antiserum and microgram quantities of thyrocalcitonin, calmodulin, gastrin, cholecystokinin, vasoactive intestinal polypeptide, serum albumin and concentrated extract of bovine pituitary gland was insignificant. Slight cross-reactivity (6%) of the antiserum with parathyroid hormone was demonstrated. Assay sensitivity was .25 ng/ml and intraassay and interassay coefficients of variation ranged from 4 to 11% and 10 to 24%, respectively. The CaBP immunoreactivity was not affected by endogenous Ca concentrations. Plasma and serum concentrations of immunoreactive CaBP were similar. The CaBP concentrations were unaffected when coagulated and anticoagulated blood samples were stored at 4 or 22 C for up to 72 h and when serum was stored at -20, 4 or 22 C for 8 d. Serum CaBP concentrations in cattle were not affected by gonadal steroids, but may have been influenced by age. Treatment with 500 mg of vitamin D3, but not 50 mg of dihydrotachysterol, significantly increased serum Ca and CaBP concentrations in Holstein heifers after a lag period of 7 to 10 d. Serum Ca and CaBP concentrations began to increase in serum at approximately the same time and both exhibited parallel responses to treatment with vitamin D3. Serum Ca concentrations were positively correlated (r = .81) with CaBP concentrations and this relationship was described by the equation, Y = 6.85 + 1.01X - .03X2. Serum Ca and CaBP concentrations were still elevated in heifers 75 d after initial treatment with vitamin D3. The radioimmunoassay we describe provides an opportunity to investigate the role of CaBP in Ca homeostasis during growth, pregnancy, lactation, parturient paresis and other physiological and pathological states in cattle.

Pituitary immunoreactive calcitonin-like material: Lack of evidence for cross-reactivity with pro-opiomelanocortin

In mammals, calcitonin (CT) is synthesized, stored, and secreted by intrathyroidal C cells. Several reports have suggested the presence of immunoreactive CT in the pituitary gland. We have studied the rat pituitary gland using a radioimmunoassay for CT and have also found immunoreactive CT-like material. Assay of extracts of whole rat pituitary glands was performed using a radioimmunoassay for human CT, which gave identical dilution curves with synthetic human CT (hCT), synthetic rat CT (rCT), and mouse and rat thyroid extracts, but not with a variety of other pituitary and hypothalamic peptides. Immunoreactive CT (iCT) content of extracts of whole pituitary glands ranged from 6 to 72 pg/mg wet weight of tissue (60–840 pg/whole pituitary gland), whereas iCT was not measureable (less than 5 pg/mg tissue) in similar extracts of hypothalamus and cerebral cortex. Gel filtration studies of pituitary extracts showed a peak of iCT, which eluted with 125I-rCT and diluted in parallel with rCT. To investigate whether the pituitary iCT was related to pro-opiomelanocortin, extracts of ACTH-producing AtT20 D16 cells from mice, which contain the ACTH precursor in large quantities, were examined and no iCT was found. Immunohistochemical studies of rat pituitary glands with peroxidase-antiperoxidase and immunofluorescent techniques showed positive staining for CT in cells in the pars anterior, but not in the pars intermedia or pars nervosa; this staining was not eliminated when the antiserum was absorbed with CT under conditions that completely obliterated staining of rat thyroid glands. Double staining demonstrated essentially two distinct populations of cells, one positive for CT and another positive for ACTH, with less than 1% of the cells positive for both ACTH and CT. Immunoreactive CT-like material was present in the pituitary glands of rats thyroparathyroidectomized 18 days before they were killed, but was diminished. Biosynthetic labeling in vitro of rat pituitary glands with 3H-leucine showed incorporation into prolactin; there was no incorporation into CT. No in vitro secretion of iCT by whole rat pituitary glands either basally or after high K + stimulation was observed. We conclude that: (1) a substance that has certain immunologic and size characteristics of CT is present in minute amounts in the pituitary gland of rats; (2) this material is not a part of the ACTH precursor; and (3) positive immunohistochemical staining in pituitary glands may not be specific for authentic CT.

Neurotensin release from rat hypothalamus in vitro.

Neurotensin is a hypothalamic peptide which inhibits secretion of TSH in the rat in vivo. We have demonstrated a calcium-dependent release of neurotensin from incubated rat hypothalamus in response to depolarizing stimuli, as well as a dose-dependent stimulatory effect of tri-iodothyronine (T3) on neurotensin secretion. We suggest that part of the neuroendocrine control of TSH secretion involves the interaction of T3, neurotensin and TSH; the presence of neurotensin in extracts of anterior pituitary gland is further evidence for its hypophysiotrophic role.

Purification of an alternate form of the alpha subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide.

Extracts of bovine anterior pituitary glands contain significant amounts of material with immunological properties similar to those of the common, alpha, subunit isolated from the pituitary glycoprotein hormones. Purification of this "free alpha-like" material and analysis show it to contain an additional site of glycosylation not present in the alpha subunit isolated from intact glycoprotein hormones. This additional oligosaccharide is O-linked to a threonine residue corresponding to threonine-43 of bovine lutropin-alpha. Carbohydrate analysis shows 1.7 mol of sialic acid, 0.8 mol of galactose and 0.9 mol of galactosamine/mol of oligosaccharide. A similar structure for the free alpha-like material as compared to bovine lutropin-alpha is evident from equal potency in an anti-lutropin-alpha radioimmunoassay, a similar amino acid composition and similar but not identical peptide maps. The free alpha-like material is distinct from lutropin-alpha in that the free alpha-material contains sialic acid and galactose, has a slightly higher apparent molecular weight, an increased negative charge, and will not reassociate with native lutropin-beta. Peptide maps of the tryptic peptides of the free alpha-like material show additional differences (other than the O-linked oligosaccharide) when compared to peptide maps of lutropin-alpha; thus additional modifications are probably present.

Rimorphin, a unique, naturally occurring [Leu]enkephalin-containing peptide found in association with dynorphin and alpha-neo-endorphin.

The tridecapeptide NH2-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-COOH has been purified from extracts of bovine posterior pituitary glands. This unique peptide, which has been given the name "rimorphin," is a major [Leu]enkephalin-containing peptide in all tissues examined that contain dynorphin and alpha-neo-endorphin. However, except for the initial hexapeptide sequence, it is structurally unrelated to the other two peptides.

Isolation and NH 2-terminal sequence of a novel porcine anterior pituitary polypeptide : Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60

An M r 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH 2-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).

Role of serum in maintenance of functional hepatocytes in primary culture.

Serum at 5 to 10% is required for maintenance of functional adult rat hepatocytes in primary culture. One effect of the serum is to induce attachment and spreading of hepatocytes on plates as monolayers. Another role is to maintain cell viability for over 2 days. For the first effect, serum could be replaced completely by fibronectin (Fn). The effects of Fn on attachment and spreading of cells were dose-dependent and maximum at 10 micrograms/ml. Cells in serum-free medium on Fn-coated dishes showed similar activities of glycogenolysis and glycogenesis to cells cultured in medium containing 5% calf serum on untreated dishes in response to glucagon, dibutyryl cyclic AMP (bt2c AMP), isoproterenol and insulin. The increase in alkaline phosphatase [EC 3.1.3.1] activity and induction of tyrosine aminotransferase [EC 2.6.1.5] by dexamethasone (Dex) in cells under the two conditions were also similar. However, the inductions of tryptophan oxygenase [EC 1.13.11.11] by Dex, glucagon, and bt2cAMP were 4-7 times higher in cells cultured in serum-free medium. The inductions by Dex plus glucagon in the two types of cultures were inhibited similarly by insulin. In serum-free medium containing Dex and insulin in Fn-coated dishes, the cells survived as monolayers for about 50 h without detachment from the dishes, but for longer survival it was necessary to add 5% serum to the medium. A fraction with a molecular weight of over 50,000 from serum was separated by ultrafiltration and this fraction showed a similar effect to serum in increasing survival. A similar factor, but with about 70 times higher specific activity, was found in an extract of bovine pituitary gland.

Characterization of rimorphin, a new [Leu] enkephalin-containing peptide from bovine posterior pituitary glands

A previously unidentified [Leu] enkephalin-containing tridecapeptide has been purified from extracts of bovine posterior pituitary glands. The peptide, which we have named rimorphin, has the sequence NH 2-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-VaI-Thr-COOH. It is a major enkephalin-containing peptide in the bovine posterior pituitary gland and is highly active in the guinea pig ileum assay.

Molecular composition of human luteinizing hormone: biological and immunological profiles of highly purified preparations after electrofocusing.

Five highly purified human pituitary LH preparations (candidate preparations for a new International Reference Preparation (IRP) and coded A to E) and three highly purified commercial preparations (referred to as Kabi I, II and III) were fractionated by electrofocusing on a sucrose density gradient with ampholytes in the pH range of 3·5–10·0 The LH activity was monitored in each of the eluted fractions by an in-vitro bioassay and a radioimmunoassay procedure and the profiles of biological activity and immunoreactivity were compared with those of the first IRP of Human Pituitary Luteinizing Hormone for Immunoassay (code no. 68/40). The overall recovery of the biological activity after electrofocusing of the nine preparations ranged between 75 and 92% (mean 86%) and was higher (P < 0·05) than that of the immunological reactivity which varied between 71 and 94% (mean 79%). The profiles of the biological activity and immunological reactivity were in close agreement with each other in all preparations. Significant differences were, however, observed in the distribution of various molecular species between the currently used standard and the eight other highly purified preparations. The ratios of biological activity (B) to immunoreactivity (I) of preparation B and of the three Kabi preparations were significantly higher than those of the other five preparations, both before and after electrofocusing. After electrofocusing, a slight but significant (P < 0·05) rise in the B/I ratios was observed, suggesting that some biologically inactive immunoreactive material was removed during fractionation. All purified preparations lacked the characteristic 'acidic' human LH species which accompanies FSH and which is abundant in the IRP (69/104) and is also present in aqueous extracts of postmenopausal pituitary glands. A comparison of the electrofocusing profiles of the nine highly purified preparations with those of human LH from plasma and individual pituitary glands revealed marked differences, suggesting that the methods used for the purification of the hormone significantly altered its molecular composition.

Growth hormone and the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells are induced or enabled to undergo adipose conversion in the presence of an extract of pituitary gland. Adipogenic activity is found in standard growth hormone preparations derived from different species. Further purification of rat growth hormone by several methods does not remove its adipogenic activity. Human growth hormone synthesized in Escherichia coli is also effective. Adipogenic activity is not associated with other pituitary polypeptides. Since growth hormone acts on preadipose cells in the absence of any other cell type, a mechanism exists for the direct participation of the pituitary gland in the regulation of this form of mesenchymal differentiation.

Changes in the plasma concentrations of luteinizing hormone, progesterone, oestradiol and testosterone and in the binding of follicle-stimulating hormone to the theca of follicles during the ovulation cycle of the hen (Gallus domesticus).

The changes in the binding of FSH during follicular maturation were examined in the hen using 125I-labelled bovine FSH (bFSH) and unlabelled bFSH. The binding of 125I-labelled bFSH was not inhibited by bovine LH or chicken LH but was inhibited by extracts of chicken pituitary glands. The ovarian stroma, which contained both interstitial tissue and small follicles, bound the greatest amount of FSH. As the follicles progressed through the yolk-filled hierarchy of maturation, they bound decreasing amounts of FSH. In the two largest follicles of the hierarchy, there was a significant increase in the binding of FSH 12-16h before ovulation. There were two peaks in the concentrations of LH; a preovulatory peak occurred 4-6h before ovulation and a second peak occurred 14-16h before ovulation. Plasma concentrations of testosterone, oestradiol and progesterone began to rise 9, 8 and 6h, respectively, before ovulation. These data are consistent with the hypothesis that changes in the gonadotrophin concentration and binding regulate the order of the follicular hierarchy and the onset of preovulatory steroidogenesis in the hen.

Studies on the biological and immunological properties of human follitropin: profiles of two international reference preparations and of an aqueous extract of pituitary glands after electrofocusing.

Abstract. Two international reference preparations, the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH/ICSH) for Bioassay (identified hereafter by its code no.: 69/104) and the First International Standard of Urinary FSH and LH (ICSH) for Bioassay (hereafter: hMG 1st IS) and an aqueous extract of human pituitary glands (hPE) were fractionated in triplicate by isoelectric focusing on sucrose density gradient (Ampholine, pH range 2.5–10.0). The FSH activity was monitored in each fraction by an in vitro bioassay, a radioimmunoassay and a receptor binding assay. Unfractionated 69/104 was used as standard in each assay system. Compared with the hPE, the activity profiles of the 69/104 and hMG reference preparations were spread over a significantly wider pH range; the biological activity eluted in the pH range of 3.5–5.0 was of the order of 90% for hPE, 72% for 69/104 and less than 60% for hMG. Major variations in biological to immunological (B/I) and biological to receptor binding (B/R) activity ratios were observed in the individual electrofocusing fractions. The B/I ratios ranged from 0.8 to 2.2 (69/104), 1.0–5.7 (hMG) and 1.3–6.0 (hPE), respectively. The variation in the corresponding B/R ratios was: 0.5–1.7 (69/104), 0.58–4.0 (hMG) and 0.55–1.5 (hPE). When equal aliquots from each of the electrofocusing fractions were combined and this 're-constituted' pool was compared with the starting material, significant differences were observed in the B/I ratios: 1.63 instead of 1.0 (69/104), 2.58 vs 2.34 (hMG) and 2.72 vs 1.32 (hPE). There was no significant change in the mean B/R ratios before and after electrofocusing: 1.0 vs 1.02 (69/104), 1.09 vs 1.02 (hMG) and 1.05 vs 1.04 (hPE). The mean recovery of biological activity in each of the three reconstituted pools of the three preparations was 97% for 69/104, 88% for hMG and 98% for hPE, and the corresponding recoveries of receptor binding activities were 95%, 94% and 98%, respectively. In contrast, the mean recovery of immunological reactivity was only 55% (69/104), 80% (hMG) and 47% (hPE), respectively. The reduction in the immunological reactivity of the 69/104 preparation without any apparent loss of biological or receptor binding activities following electrofocusing indicates that its immunoreactivity is unstable under mild experimental conditions, which do not influence its biological and receptor binding activities. Hence, whereas this preparation might possibly be suitable as a standard for in vitro bioassays and receptor binding assays, it is unsuitable as a standard for the radioimmunoassay of hFSH.

Immunoreactive calcitonin in the central nervous system of the pigeon

To determine whether calcitonin (CT) is present in avian central nervous system (CNS), as it has been recently found in the CNS of other species, we studied extracts of pigeon ( Columba livia) brain by immunochemical methods. A sensitive and specific radioimmunoassay for salmon CT was used together with gel filtration and high performance liquid chromatography (HPLC). Extracts of hypothalamus contained 31.5 ± 9.65ng/g wet weight as salmon CT-like immunoreactivity while extracts of midbrain and brain stem contained 17.0 ± 5.00and2.85 ± 2.1ng/g wet weight respectively. Extracts of whole pituitary gland, other areas of the CNS and various peripheral tissues were below the detection limits of our assay, but salmon CT- like immunoreactivity was present in ultimobranchial gland (UBG), thyroid gland and lung tissue extracts. On Sephadex G-50 chromatography extracts of hypothalamic, midbrain, UBG and thyroid gland gave an immunoreactive peak which co-eluted with synthetic salmon CT. However, on HPLC the material in pigeon hypothalamus,. midbrain, UBG and thyroid gland eluted in an identical manner when compared to each other but the major peak was 2 ml after that of synthetic salmon CT. These results clearly show that a salmon CT-like immunoreactive molecule is present in pigeon hypothalamus and midbrain, in addition to its main locations in the UBG and thyroid gland.