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In most previous reports material with corticotrophin releasing factor (CRF) activity has been obtained from hypothalami after extraction with dilute aqueous acid. Such conditions allow substantial proteolytic degradation. By adopting conditions designed to precipitate proteases and by using information on the nature of CRF gained from earlier studies, rapid large scale extraction and partial purification of porcine hypothalamic CRF in high yield was achieved. After extraction with 0·2 m-HCl: acetone (1: 1, v/v), centrifugation and ultrafiltration, considerable preliminary purification of the CRF activity was achieved by adsorption onto carboxymethylcellulose and subsequent elution at increased salt concentration. Following ion-exchange chromatography of the extract on carboxymethylcellulose, CRF activity was obtained in good yield (minimal effective dose of about 1–2 μg/ml) for ACTH release in an in-vitro CRF bioassay utilizing a coupled isolated pituitary cell–adrenal cell system. The data indicated that the previously reported heterogeneous corticotrophin releasing factors of low activity may be a consequence of proteolytic degradation.
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The inhibitory action of porcine follicular fluid (FF1) on the spontaneous maturation of isolated rat oocytes was studied. Both FF1 and a low-molecular-weight fraction thereof (PFF1) inhibited the maturation of oocytes in culture. When the oocytes were scrutinized for maturation after culturing for 6 h in a medium containing 50% (v/v) FF1, the incidence of germinal vesicle breakdown (GVB) was reduced from 75 to 53% (P < 0·001) in oocytes collected 20 h after administration of pregnant mare serum gonadotrophin (PMSG), and from 94 to 53% (P < 0·01) in oocytes collected from preovulatory follicles 44 h after treatment with PMSG. Prolongation of the culture period to 20 h resulted in an increase in the number of oocytes undergoing GVB to 58% (harvested 20 h after PMSG treatment; P < 0·001) or 71% (44 h after treatment with PMSG; P > 0·05). Inhibition of GVB by PFF1 occurred at concentrations ≥ 0·085 mg protein/ml; the extent of inhibition was related directly to the concentration of inhibitor and inversely to the duration of culture and the developmental stage of the follicles from which the oocytes were derived. The inhibition of the resumption of meiosis by FF1 or PFF1 was overcome by ovine LH (5 μg/ml). However, GVB was delayed even in the presence of LH, compared with controls cultured in the absence of the inhibitor. These results demonstrate that the maturation-inhibiting action of porcine follicular fluid is not species specific and that the sensitivity of the rat oocyte to the inhibitor changes during the course of follicular development.
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The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 μg ovine FSH pretreated with antiserum to LH or 10 μg human FSH resulted in an 11- to 18-fold, five-to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1–2 mm), medium (3–5 mm) and large (6–12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles.
During incubation periods of 15 min, 1·0 μg ovine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1·0 μg ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation.
It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay.
Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
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Kumar R, Yang K, Vallet JL & Christenson RK 2003 11β-Hydroxysteroid dehydrogenase and glucocorticoid receptor messenger RNA expression in porcine placentae: effects of stage of gestation, breed, and uterine environment. Biology of Reproduction 69
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ABSTRACT
Immunoreactive α-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1–26 amino acid sequence of the N-terminus of the α chain of porcine inhibin (p1–26 α-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1–26 α-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1·15–1·21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M r 45 000) which immunoblotted against p1–26 α-inhibin antibody.
In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1–26 α-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate α-inhibin material, and immunocytochemical localization studies of α-inhibin in porcine and ovine luteal sections were negative.
Our results are consistent with the intracellular packaging/storage of a form of α-inhibin (M r similar to that of α-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for α-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.
Journal of Endocrinology (1992) 134, 341–352
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ABSTRACT
12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumour promoter and shows several biological activities of epidermal growth factor (EGF). EGF and TPA stimulated proliferation and inhibited differentiation of porcine thyroid cells in primary culture. They also stimulated [3H]thymidine incorporation and inhibited an early step in thyroid hormone synthesis (iodine organification). The results indicate that EGF and TPA switch the developmental course of porcine thyroid cells from differentiation to proliferation.
J. Endocr. (1987) 113, 485–487
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Culture medium 199 supplemented with follicular fluid from 1–2 mm antral porcine follicles inhibited spontaneous luteinization of granulosa cells from preovulatory porcine follicles in vitro. Three characteristics of luteinization were inhibited: morphological transformation, progesterone secretion, and accumulation of cyclic AMP in response to LH. The last was inhibited more effectively by culture media containing 50% follicular fluid than by media containing 20% follicular fluid. The inhibitory actions of the follicular fluid were not altered by charcoal or petroleum ether extraction. Follicular fluid from large follicles (6–12 mm) did not exhibit any of these inhibitory actions. These observations may indicate the presence of a luteinization inhibitor in the fluid of small follicles which (1) is lost by the time the follicle reaches the preovulatory stage, or (2) is overcome by a stimulatory agent which may accumulate as the follicle grows.
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ABSTRACT
Thyrocytes isolated from porcine thyroids by mechanical and enzymatic dispersion and cultured in Eagle's minimal essential medium, supplemented with 5% (v/v) fetal calf serum, glutamine and cortisol, formed a continuous monolayer within 48 h. This monolayer was without cytochemical peroxidase and diaphorase (NADPH reoxidation) activity. In the presence of bovine thyrotrophin (bTSH; 50 mu./l) the cells developed a follicular-like architecture which was maximal at 4 days before reverting back to a uniform monolayer at 6 days. There were no detectable changes in the total DNA content over this period. The follicular structures had marked diaphorase and peroxidase activity, the latter being apically distributed. Concomitant with follicle formation bTSH induced uptake and organification of iodide presented to the cells during the last 6 h of culture. The extent of this process depended on the dose of bTSH and the duration of stimulation. The most sensitive effects for both iodide uptake and organification occurred with 1 mu. bTSH/1 and were maximal with 100 mu./l. Uptake and organification were increased 20±8-fold and 9·6±2- fold (n = 10) respectively over the control with 100 mu./l and the doses of bTSH at which a half maximal response was seen (ED50) were 15±2 and 7±1 (s.d) mu./l (n = 10) respectively.
On changing the culture medium to a serum-free system using HB101 culture medium the stimulation time for the most sensitive bTSH effect was reduced to 2·5 days. Moreover the sensitivity of iodide uptake to bTSH was dramatically increased being significantly stimulated by 0·1 mu./l, saturated with 10 mu./l, and having an ED50 of 7 ± 0·2 mu. bTSH/1 (n = 7). Over the dose range 0·1–10 mu. bTSH/l intra- and interassay coefficients of variation were 7%±1·5 (n = 15) and 11% ± 2·5 (n = 15) respectively. Cyclic AMP production in cultures incubated under similar conditions (i.e. after chronic TSH stimulation) was also stimulated by bTSH doses in the range 15·6–125 mu./l. Cells stimulated for 2·5 days with dibutyryl cyclic AMP (range 1 μmol/1–2 mmol/l) also actively concentrated iodide in a dose-dependent fashion.
The presence of normal human serum in the medium yielded a progressive and dose-related decrease in TSH-stimulated iodide uptake. This inhibitory effect occurred with serum concentrations as low as 0·1%.
In conclusion, a porcine cell culture system is described in which iodide uptake is significantly stimulated by 0·1 mu. bTSH/l, a sensitivity inferior only to that reported with the cytochemical bioassay.
J. Endocr. (1985) 106, 13–20
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Abstract
Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a K d of 0·45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake. These results are compatible with the notion that ET-1 negatively regulates TSH-induced iodide uptake in an autocrine manner.
Journal of Endocrinology (1994) 142, 463–470
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ABSTRACT
The effects of insulin-like growth factor-I (IGF-I) on cytoplasmic pH (pHi) and [3H]thymidine incorporation were studied in primary cultures of porcine thyroid cells. IGF-I alkalinized thyroid cells and stimulated thymidine incorporation in a dose-dependent manner; the effects of IGF-I on alkalinization (the maximal rates of change of cytoplasmic pHi/min ((dpHi/dt)max)) and thymidine incorporation were observed at 2 ng/ml and were maximal at 100 ng/ml, with half-maximal stimulation at approximately 10 ng/ml. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of IGF-I on thyroid cell proliferation. Several mitogens and comitogens which activate sodium hydrogen exchange, including epidermal growth factor, platelet-derived growth factor and nerve growth factor, have been listed. Activation with IGF-I has not, however, been presented before. Thus the present study constitutes the first demonstration of IGF-I-stimulated activation of Na+/H+ exchange or cell alkalinization.
Journal of Endocrinology (1990) 127, 305–309
