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ABSTRACT
Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate 125I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0·1–0·3 mU/ml. Addition of ClO4 − to the basal medium inhibited transcellular transport, whilst addition of ClO4 − to the apical medium was much less effective.
Constituents labelled with 125I in the apical medium were analysed. The amount of protein-bound 125I measured by acid precipitation was 3–8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate–polyacrylamide gel electrophoresis revealed that most of the 125I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium.
The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential.
Journal of Endocrinology (1990) 126, 275–281
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ABSTRACT
Thyrotrophin stimulated release of radioiodine from the organic iodine pool of cultured porcine thyroid cells. The response was well developed within 2–4 h of incubation. Inhibition of the Na+/K+ pump with ouabain or incubation in sodium-free medium inhibited the response to TSH. The magnesium content of cultures was reduced by ouabain, and increasing the magnesium concentration of the medium to 10 mmol/l reversed the inhibition of the TSH response by ouabain. After prolonged incubation (4–6 h), ouabain in magnesium-enriched medium stimulated release of radioiodine. Its effects were not additive with those of TSH. Incubation for 4–6 h in media of reduced sodium concentration (34 mmol/l) also stimulated release. Sodium-free medium alone did not alter basal release rates, but magnesium enrichment of sodium-free medium promoted release after 4 h of incubation. It was concluded that the previously reported inhibition of the TSH response in thyroid tissue by ouabain or sodium-free medium was due to secondary derangements of cellular function rather than to a specific blockade of the secretomotor signal. The data are consistent with the hypothesis that a reduction in the sodium ion electrochemical gradient across the cell membrane mediates the secretomotor effect of TSH.
J. Endocr. (1987) 112, 399–405
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ABSTRACT
Cultured porcine thyroid cells did not reassociate into functional follicles in the presence of TSH unless the initial seeding density was adequate. At 0·2 × 106 cells/35 mm diameter culture dish the cells rapidly formed a monolayer even in the presence of TSH (128 μu./ml), and radioiodide uptake was not significantly increased compared with that in control cells. Seeding densities of 1–3 × 106 cells/dish resulted in cultures which responded to TSH with follicular development and increased radioiodide uptake. A cell-free membrane fraction of thyroid homogenate restored the ability of cultures seeded at low densities to respond to TSH with development of follicular morphology and increased radioiodide uptake. Delaying the addition of TSH by 48 h markedly reduced the stimulation of follicular development and radioiodide uptake of cultures. Addition of membrane fractions, or an alkali-soluble fraction of membranes, at zero time improved the responses to TSH added after a 48-h delay. It was concluded that maintenance of differentiation and of TSH-responsiveness in cultured thyroid cells was influenced by cell–cell contact.
J. Endocr. (1987) 113, 223–229
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To examine the manner in which the FSH surge of one oestrous cycle recruits follicles for ovulation in the subsequent cycle, porcine follicular fluid (PFF) was used to alter the pattern of endogenous FSH secretion during the periovulatory period. Ovaries of animals killed at oestrus or metoestrus were examined histologically for the presence of large follicles (greater than 400 μm in diameter) after treatment. Large follicles were absent in ovaries of all PFF-treated animals at oestrus, while control rats had an average of 2·7 large follicles per ovary. By metoestrus, however, ovaries of rats treated with PFF contained several large, healthy follicles. Only when PFF treatment was continued throughout the evening of oestrus was the appearance of large follicles prevented at metoestrus.
Our results suggest that the prolonged oestrous portion of the FSH surge, rather than the pro-oestrous portion, is responsible for follicular recruitment during the normal oestrous cycle in the rat. They also indicate that compensatory follicular development occurs in response to the FSH rebound which has been shown to follow FSH suppression.
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20α-Hydroxypregn-4-en-3-one (20α-dihydroprogesterone) has been isolated from porcine ovaries under in vitro conditions (Bjersing & Carstensen, 1967; Cook, Kaltenbach, Norton & Nalbandov, 1967; Schomberg, 1967). No 20β-epimer has hitherto been demonstrated in the pig.
While the progesterone concentration was being investigated in the corpora lutea of the pregnant gilt using a method similar to that of Rowlands & Short (1959), two u.v. absorbing substances were found on the paper chromatograms with Rf values identical to authentic 20α- and 20β-hydroxypregn-4-en-3-one (20α-ol and 20β-ol) These substances were eluted, and gave the same response as the authentic steroids when examined by spectrophotometry, namely an absorption maximum in ethanol at 240 mμ, an absorption maximum at 285 mμ on heating with cone. H2SO4, and 20α-ol gave an absorption maximum at 485 mμ when treated with sulphuric acid:80% ethanol (Wiest, 1959). Both substances were acetylated with 0·1 ml. pyridine and acetic anhydride
Lactation and Mammary Gland Biology Group, Department of Animal Science, US Meat Animal Research Center, Agriculture and Agri-Food Canada, Department of Physiology, Department of Animal Science, Department of Animal Science, The University of Vermont, 570 Main Street, Burlington, Vermont 05405, USA
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Lactation and Mammary Gland Biology Group, Department of Animal Science, US Meat Animal Research Center, Agriculture and Agri-Food Canada, Department of Physiology, Department of Animal Science, Department of Animal Science, The University of Vermont, 570 Main Street, Burlington, Vermont 05405, USA
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Lactation and Mammary Gland Biology Group, Department of Animal Science, US Meat Animal Research Center, Agriculture and Agri-Food Canada, Department of Physiology, Department of Animal Science, Department of Animal Science, The University of Vermont, 570 Main Street, Burlington, Vermont 05405, USA
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concentrations of PRL in serum between d 90 and 109 of gestation ( Farmer & Petitclerc 2003 ). Postpartum suppression of serum PRL also impairs lactation and the growth of nursing pigs ( Farmer et al . 1998 ). Conversely, administering porcine pPRL to lactating
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Abstract
In this study, the epitope of a murine PS-7·6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70–95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7·6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7·6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70–95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75–90) was the optimal sequence recognized by PS-7·6 mAb. Sequential alanine substitution of each residue of pGH(75–90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7·6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75–95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i+3 (i+7) of the critical amino acids appears consistent with PS-7·6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7·6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals.
Journal of Endocrinology (1995) 145, 169–174
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ABSTRACT
In cultured porcine and human thyroid cells, electrophysiological and morphological studies showed that cultivation in the presence of TSH, prostaglandin E2 (PGE2) or dibutyryl cyclic AMP (dbcAMP) maintained normal cell polarity with iodine incorporation and organification. Cells cultivated in the absence of these substances had inverted cell polarity and lacked iodide incorporation.
In the presence of TSH, PGE2 or dbcAMP, the thyroid cells formed follicles with normal cell polarity and the microvilli pointed toward the follicle lumina. Intracellular resting potentials were − 60 mV and the electrical potentials in the follicle lumina were negative at − 20 mV. The transmembrane potential differences (p.d.) between the follicle lumina and the epithelial cells were − 40 mV and those between the epithelial cells and the culture media − 60 mV.
In the absence of TSH, PGE2 or dbcAMP, the thyroid cells formed 'domes' or hollow spheres with inverted cell polarity and the microvilli pointed toward the culture media. Intracellular resting potentials were − 40 mV, being less negative than those in the presence of TSH, PGE2 or dbcAMP. The electrical potentials in the 'dome' or hollow sphere cavities were positive at + 19 mV. The transmembrane p.d. between the culture media and the epithelial cells was − 40 mV and that between the epithelial cells and the cavities − 60 mV, indicating that electrophysiologically the cell polarity was inverted in the absence of TSH, PGE2 or dbcAMP.
No significant differences in electrophysiology and iodine metabolism were observed between normal and Graves' human thyroid cells in culture.
J. Endocr. (1984) 101, 189–196
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Abstract
This study examined the effects of tumour necrosis factor-α (TNFα) on basal and stimulated progesterone secretion, as well as prostaglandin F2α production, by small, large and mixed porcine luteal cells and assessed the action of TNFα in the presence and absence of indomethacin. Corpora lutea were isolated from gilts on days 8–9 of the oestrous cycle and enzymatically dissociated. Luteal cells were either subjected to elutriation to isolate small and large cells or were separated from erythrocytes by a polysucrose gradient to serve as the mixed luteal cell group. Then 24-well culture plates were seeded with 150 000 small, 30 000 large and mixed (30 000 large+100 000-250 000 small) luteal cells suspended in 1 ml medium 199 media supplemented with 5 μg insulin/ml, 40 ng cortisol/ml and 50 μg low-density lipoproteins/ml. Cells were cultured for up to 24 h in a humidified incubator at 37 °C with 5% CO2 in air. TNFα time- and dose-dependently suppressed (P<0·05) LH-induced, but not basal, progesterone secretion by small luteal cells. Moreover, TNFα inhibited (P<0·05) forskolin-mediated, but not cyclic AMP-mediated, progesterone secretion by small luteal cells. The LH-stimulated progesterone secretion by small luteal cells was not affected by TNFα in the presence of indomethacin. Progesterone secretion by large and mixed luteal cells was not affected by TNFα. Prostaglandin F2α production by small and mixed, but not large, luteal cells was enhanced (P<0·05) by TNFα. These data demonstrated that TNFα acts primarily on a target cell(s) in the small cell fraction, and the cytokine-induced inhibition of LH-stimulated progesterone secretion by small cells was mediated by prostaglandin F2α and involved a site(s) proximal, but not distal, to cyclic AMP generation.
Journal of Endocrinology (1994) 143, 75–83
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ABSTRACT
The cytoplasmic concentration of free calcium was measured using aequorin, a calcium-sensitive photoprotein. The Ca2+ ionophore A23187 induced a rise in cytoplasmic free calcium and iodide discharge in cultured porcine thyroid cells. The minimum dose of A23187 effecting an increase in cytoplasmic free calcium induced iodide discharge. The A23187-induced rise in cytoplasmic free calcium was followed by iodide discharge. The results indicate that A23187-induced iodide discharge is mediated by a rise in the cytoplasmic concentration of free calcium.
J. Endocr. (1987) 115, 477–480
