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Steroid hormone concentrations (progesterone, oestrone, oestradiol-17β) in porcine follicular fluids were measured by radioimmunoassay. In confirmation of several previous studies, as follicular maturation proceeded, the concentrations of all three steroids increased, although the relative amounts of the oestrogens decreased compared to progesterone. Analysis of fluids for steroid-binding components was undertaken using ion-exchange chromatography, gel filtration and steroid-binding assays. Evidence is presented in support of a labile binding component which exhibits high affinity and low capacity for progesterone and little or no affinity for oestradiol. Our findings are discussed in relationship to the known retention of progesterone within preovulatory follicles and to previous reports of steroid-binding components in follicular fluids.
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SUMMARY
Pig posterior pituitary lobe powder contains a peptide structurally identical to the 18–39 portion of porcine adrenocorticotrophin (ACTH). Its extraction by several different procedures is described and its susceptibility to degradation in the presence of 5% acetic acid has been noted. This degradation has been ascribed to the presence of acid proteases in the acetone-dried powder of the posterior pituitary lobe. The peptide has been isolated by chromatography on Biogel P6 and DEAE-cellulose, and characterized by enzyme fragmentation studies. It resembles the peptide isolated from rat neurointermediate lobes termed 'corticotrophin-like intermediate lobe peptide' and it is suggested that it is derived by the intracellular cleavage of ACTH in the pars intermedia cells, with subsequent formation of α-melanocyte-stimulating hormone from the other adrenocorticotrophic fragment.
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Isolated porcine thyroid cells were surface-labelled with 125I using the lactoperoxidase technique. Samples of the cells were then cultured and harvested at various intervals for up to 7 days. The labelled proteins remaining on the cells or shed into the culture medium were analysed by electrophoresis on polyacrylamide gels run in sodium dodecyl sulphate. These studies indicated that the several different surface proteins of the thyroid cells were lost from the cell surface at similar rates (half-time of approximately 28 h) as the result, at least in part, of a process which depended on active cell metabolism. In addition, the gel profiles obtained from analysis of both medium and membrane-bound labelled proteins were similar and this suggested that peptide cleavage was not involved in the shedding of the majority of these proteins.
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Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 microM haemin (a haem oxygenase substrate) was approximately 1.7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.
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ABSTRACT
The membrane potential of cultured porcine thyroid follicular cells depolarized by up to 20 mV from the resting value of about − 73 mV on exposure to β-adrenoceptor agonists. A similar response was induced by TSH or dibutyryl cyclic AMP. α-Adrenoceptor agonists were without effect. The receptor subtype was shown to be (at least predominantly) β2 by the order of potency for β-agonists (isoprenaline ≅ fenoterol⪢adrenaline >noradrenaline) and by the relative potency of selective β-antagonists (ICI 118,551 ⪢atenolol). The α-agonist phenylephrine had no effect on the TSH response but weakly inhibited the β-agonist response. Rather than a physiological antagonism between α- and β-adrenoceptor-mediated responses, this effect was shown to be due to the weak β-antagonist effect of phenylephrine since the α-antagonist phentolamine failed to potentiate the depolarizing response to the mixed agonist nor-adrenaline, and also failed to block the inhibitory action of phenylephrine on the β-agonist effect. Sensitivity to β-agonist was enhanced by omission of serum from the culture medium and reduced by exposure to β-agonists or a high concentration of TSH or dibutyryl cyclic AMP.
J. Endocr. (1985) 107, 23–30
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ABSTRACT
Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment from the culture dish substrate (domes). The transepithelial potential (TEP), positive on the basal side, was 12·9 ± 0·4 (s.e.m.) mV (n = 93) under control conditions, increasing to 38·9 ± 0·3 mV (n = 281) when fluid transport was stimulated by prostaglandin E2 (PGE2; 1 μmol/l). Forskolin (1 μmol/l) and 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate (0·5 mmol/l) were also effective in increasing TEP. Addition of amiloride in concentrations sufficient to block fluid transport (100 μmol/l) reduced the TEP to 5·8 ± 0·3 mV (n=76). Substitution of N-methyl-d-glucamine for sodium in the medium reduced the PGE2-stimulated TEP to 13·4 ± 0·8 mV (n = 32). Substitution of gluconate for chloride increased the TEP to 40·3 ± 0·4 mV (n = 160). Removal of bicarbonate or potassium from the medium, or addition of ouabain (200 μmol/l) were also effective in reducing the TEP. In media of low bicarbonate concentration (1 mmol NaHCO3/l), acetazolamide (1 mmol/l) reduced the TEP. Fluid transport by the monolayer as measured by the change in height of domes was increased by PGE2 (1 μmol/l). PGE2-stimulated fluid transport was inhibited by sodium or chloride ion substitution, bicarbonate removal or the addition of ouabain (200 μmol/l) or amiloride (100 μmol/l). It was concluded that fluid transport in thyroid monolayers is mediated by rheogenic sodium transport with chloride transport being passive, electrogenically coupled to sodium transport. Sodium entry to the apical pole of the cells occurs by an amiloride-sensitive mechanism, and sodium extrusion at the basal pole depends on the Na+/K+ ATPase.
J. Endocr. (1988) 119, 309–314
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Porcine relaxin produced a rapid, dose-related rise of cyclic AMP values in rat uterine tissue incubated in vitro. In time-course experiments, peak cyclic AMP concentrations were observed in the uterine slices at 5 min; subsequently the values fell, at first rapidly and then more slowly with the tissue concentration remaining significantly raised at 15 min. Levels of cyclic GMP in the same tissue slices were not significantly altered by relaxin. Furthermore, no increase in basal cyclic AMP values was measured in control slices prepared from the rat heart or jejunum. An increase in cyclic AMP concentration comparable to that found in the rat uterus was observed in slices of porcine uterus and cervix but not of vagina when they were stimulated with porcine relaxin. Our results suggest that the hormonal action of relaxin on the uterus and cervix is mediated through receptors linked to the enzyme, adenylate cyclase.
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ABSTRACT
Since porcine islets are considered a likely tissue source for islet transplantation we have studied the insulin secretory responses to stimuli and some of the cell surface antigen characteristics of porcine islet cells.
In a static incubation system, the threshold level of glucose required for the stimulation of insulin secretion from freshly isolated porcine islets was found to be between 2·8 and 4·2 mmol glucose/l. Arginine (5 mmol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l) potentiated insulin release induced by 8·3 mmol glucose/l. Leucine (5 mmol/l) initiated release in the presence of 2 mmol glucose/l. Neither β-hydroxybutyrate (10 mmol/l) nor octanoate (5 mmol/l) potentiated insulin release induced by 8·3 mmol glucose/l, but β-hydroxybutyrate initiated release in the presence of 2 mmol glucose/l while octanoate did not.
A 125I-labelled protein A binding assay and an enzyme-linked immunosorbent assay system were used to detect antibody binding to islet and non-islet cells. Monoclonal antibodies raised against intact rat islets were shown to bind to both porcine and rat islet cells but not to rat hepatoma tissue culture cells or rat insulinoma cells. The serum from recently diagnosed type I diabetics was shown to bind to rat islet cells in a 125I-labelled protein A binding assay, while serum from control subjects showed little, if any, binding. Porcine islet cells were unable to distinguish between the sera of recently diagnosed type I diabetics and controls in a similar assay.
In conclusion, porcine islets respond to many of the major insulin secretagogues to which human islets are sensitive. However, they have a lower secretory response to these compounds compared with that reported for human islets. Monoclonal antibody and human sera binding studies show that rat and porcine islets share some cell surface antigens but it remains to be seen whether the inability of porcine islets to discriminate between diabetic and non-diabetic sera is an advantage for transplantation, indicating less likelihood of an islet-specific autoimmune-like humoral attack.
Journal of Endocrinology (1990) 126, 43–4
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SUMMARY
Purified porcine and salmon calcitonin and synthetic salmon and human calcitonin were infused at the rate of 20 MRC mu./kg/h over a 96 h period in intact male lambs. Every calcitonin preparation significantly increased the urinary excretion of inorganic phosphorus, sodium, potassium and calcium, while the urinary excretion of magnesium was always significantly inhibited. Similar significant effects were observed with purified porcine calcitonin in thyroparathyroidectomized lambs supplemented with thyroxine. Oxidation of calcitonin with performic acid completely abolished its effects on plasma calcium and on the kidney. It is concluded that, when used at physiological doses, in sheep calcitonin has an important effect on urinary excretion of inorganic phosphorus, sodium and magnesium.
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SUMMARY
The presence of a single methionine in porcine parathyroid hormone, at position 8, permitted assessment of the role of this residue separate from the second methionine residue found at position 18 of bovine and human parathyroid hormones. Oxidation of the solitary methionine of porcine parathyroid hormone to the sulphoxide destroyed biological activity, but this was restored by subsequent reduction with cysteine. Oxidation of the hormone did not, however, affect its immunological activity; therefore, oxidation of the hormone may bring about dissociation of biological and immunological activity.
