ABSTRACT

Confluent monolayer cultures of porcine thyroid cells form dome-shaped elevations by local separation from the plastic culture dish. Formation of domes by epithelial cells in culture is generally considered to be evidence of fluid transport. A computer-controlled data acquisition system was developed to quantitate fluid transport in thyroid cultures by serial measurements of dome elevation. Thyrotrophin (10 mU/ml), prostaglandin E₂ (PGE₂; 0-01-1 μmol/l), forskolin (1 μmol/l), 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (0.5 mmol/l) and 3-isobutyl-1-methyl-xanthine (0.5 mmol/l) promoted increases in dome height over 5–120 min. Dome growth in the presence of PGE₂ (1 μmol/l) was inhibited by amiloride (0.1–100 μmol/l), ouabain (200 μmol/l), or by removal of bicarbonate and glucose from the medium. In media of reduced bicarbonate concentration (1 mmol/l compared with the control concentration of 10 mmol/l), dome growth was inhibited by acetazolamide (0.01– 1 mmol/l). These data are consistent with cyclic AMP-stimulated transport of fluid from apical to basal pole of the cells, dependent on sodium entry through the apical pole by an Na⁺/H⁺ exchanger.

J. Endocr. (1987) 115, 19-26

ABSTRACT

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Fluid transport, as measured by increase in dome height, was stimulated by prostaglandin E₂ (PGE₂; 1 μmol/l) and inhibited by amiloride (0·1–100 μmol/l). Values of the inhibition constant (K _i) with 95% confidence limits for each of a series of amiloride analogues were: 3′,4′-dichlorobenzamil (DCB), 0·090 (0·045–0·18) μmol/l; 2′,4′;-dimethylbenzamil (DMB), 0·14 (0·074–0·27) μmol/l; amiloride, 0·72 (0·33–1·8) μmol/l; 5-(N,N-hexamethylene)amiloride (HMA), 17 (5·9–43) μmol/l; 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 33 (15–71) μmol/l; and 2-guanidinobenzimidazole, 243 (110–570) μmol/l. Triaminopyrimidine was ineffective at concentrations up to 1 mmol/l. Since DCB and DMB are known to have a higher affinity for Na⁺ channels, while HMA and EIPA show higher affinity for Na⁺/H⁺ antiports, it was concluded that PGE₂-stimulated fluid transport involved an apical membrane Na⁺ channel.

Journal of Endocrinology (1989) 123, 93–97

ABSTRACT

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Stimulation of fluid transport by prostaglandin E₂ (PGE₂; 1 μmol/l) was associated with an increase in transepithelial potential (TEP). Intracellular potentials (equal to the potential difference across the apical membrane of the cell, E_apical) and the TEP were measured in individual domes so that the potential difference across the basal membrane of the cell (E_basal) could be calculated from the relationship TEP = E_apical − E_basal. The PGE₂-induced increase in TEP was associated with hyperpolarization of the basal membrane, accompanied by a slight depolarization of the apical membrane. Lines of best fit by least-squares regression showed E_apical = −20·3 mV + 0·219 TEP (correlation coefficient r = 0·627; P < 0·001) and E_basal = −20·3 mV − 0·781 TEP (r = 0·944; P < 0·001). Phenamil (1 μmol/l), a Na⁺ channel selective amiloride analogue, reduced the TEP from 13·25±0·58 (s.e.m.; n = 56) to 2·39±0·16 mV (n = 51; P < 0·001) and hyperpolarized the apical membrane potential from − 20·7±0·68 (n = 60) to −32·2±0·83 mV (n = 105; P < 0·001). The response of the TEP to phenamil was immediate, and was promptly reversed on washing; in contrast, addition of 5-(N-ethyl-N-isopropyl)amiloride (20 μmol/l; selective for Na⁺/H⁺ antiporters) resulted in a slow depolarization over 30 min with a slow recovery after washout. Exposure of the cultures to media of pH 7·04 (compared with the normal pH of 7·34) resulted in a reduced response to PGE₂, and a reduction in magnitude of E_basal. It was concluded that stimulation of ion transport by PGE₂ in thyroid monolayers involves activation of cation transport across the basal membrane.

Journal of Endocrinology (1990) 127, 197–202

Abstract

The present study has investigated an involvement of autocrine transforming growth factor-β1 (TGF-β1) in regulating the proliferative response of porcine thyroid follicular cells (TFCs) to epidermal growth factor (EGF) and TSH. Primary monolayer TFC cultures exposed to EGF over the range 0–0·4 nmol/l showed a dose-dependent increase in [methyl-³H]thymidine incorporation, whereas higher EGF doses were associated with a reduction in the level of [methyl-³ H]thymidine incorporation. TGF-β immunoneutralisation had little effect on the stimulatory action of low EGF doses, but led to an increase in [methyl-³H]thymidine incorporation at higher EGF levels. In TFC cultures exposed to TSH, the level of [methyl-³H]thymidine incorporation attained at a dose of 1 U TSH/1 was enhanced in the presence of TGF-β1 antiserum, although the similar stimulatory effect of 8-bromo cAMP was unaffected. Treatment of TFCs with phorbol 12-myristate 13-acetate (8 nmol/l) to activate protein kinase C (PKC) led to an enhanced incorporation of [methyl-³H]thymidine which was increased further after neutralisation of endogenous TGF-β1. While confirming, therefore, a role for autocrine TGF-β1 in maintaining control of TFC DNA synthesis in vitro, these findings provide evidence that an increase in the availability of autocrine TGF-β1 effected by EGF and TSH may play an instrumental role in limiting the cellular hyperplasia induced by these factors within the thyroid follicular microenvironment. Moreover, the present data also suggest that the availability of active autocrine TGF-β1 to TFCs under such conditions may be dependent upon a PKC-mediated mechanism.

Journal of Endocrinology (1996) 148, 87–94

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

ABSTRACT

The present study was designed to examine the effects of stress, associated with daily handling and placebo injection, on growth and adrenal activity in female rats. A second objective was to examine the effects of porcine GH (pGH) on growth and on serum concentrations of ACTH and corticosterone.

Treatments were administered in the morning (08.00 h) in semi-darkness, or in the evening (20.00 h) towards the end of the light period and were timed to coincide with reported periods of high and low adrenal sensitivity to ACTH.

Handling of and s.c. injection of saline into female rats (initial body weight 220 g) each morning for 21 days did not affect growth, food intake or organ weights. In contrast, daily handling and saline injection each evening caused a marked reduction in weight gain (25%), food intake (12%) and liver weight (8%), accompanied by a trend towards an increase in adrenal weight (11%), but no change in serum corticosterone concentrations.

In a second experiment, treatment of female rats (initial body weight 180 g) with pGH (5·6 mg/kg) daily at 08.00 h caused a significant improvement in weight gain (17%), but food intake and organ weights were unaltered. Daily injection of rats with pGH at 20.00 h caused a greater relative improvement in weight gain (45%) than did treatment at 08.00 h as the growth impairment caused by the stress of handling was counteracted. The adverse effect of evening injections of saline on food intake was also counteracted by pGH. Changes in liver and adrenal weights, however, were not attenuated by pGH treatment. Serum corticosterone concentrations were barely altered by pGH treatment, but serum ACTH concentrations were increased markedly (65–136%) by pGH in response to both morning and evening injection.

The results show that mild stress associated with daily handling and injection can have a profound effect on the growth of rats, but lend no support to the hypothesis that the growth impairment is mediated by corticosterone. Female rats handled and treated in the morning provide a good physiological growth model of a non-stressed animal, but rats treated in the evening are more responsive to the anabolic effects of pGH. It is suggested that pGH causes a reduction in adrenal sensitivity to ACTH and, in the absence of increased corticosterone output, high serum ACTH concentrations may contribute to part of the anabolic activity of pGH.

Journal of Endocrinology (1989) 123, 113–119

ABSTRACT

The calcium ionophore A23187 (0·1–1 μmol/l) inhibited membrane electrical polarization, uptake of ¹²⁵I, fluid transport and TSH-stimulated release of radioiodine from the organic pool in follicular cultures of porcine thyroid cells. At higher concentrations (1–30 μmol/l), A23187 promoted release of radioiodine from the organic pool. Stimulation of release of radioiodine from the organic pool by veratridine (a sodium channel agonist, 0·4–1 mmol/l) and A23187 was dependent on the calcium concentration of the medium, while TSH action was independent. Incubation in medium of very low calcium concentration (0·0177 mmol/l) resulted in enhanced release from the organic pool, which was inhibited by TSH (256 μU/ml), A23187 (25 μmol/l) or veratridine (0·5 mmol/l). These data therefore do not support the hypothesis that calcium acts as a mediator of the secretomotor action of TSH, but suggest the possibility of a TSH-induced increase in intracellular calcium as a regulatory negative-feedback mechanism.

J. Endocr. (1988) 116, 373–380

ABSTRACT

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14–4551 μg/l for MT-hGH and 23–1578 μg/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Journal of Endocrinology (1989) 120, 481–488

ABSTRACT

The origin and biosynthesis of 4-oestrene-3,17-dione (19-norandrostenedione), a major steroid in porcine ovarian follicular fluid, was investigated by culturing granulosa cells from 4–6 mm follicles of prepubertal gilts with radiolabelled androstenedione and 19-hydroxyandrostenedione. Steroid metabolites were purified by solvent extraction and lipophilic column chromatography, and analysed by C₁₈ reverse-phase high-performance liquid chromatography. 19-Hydroxyandrostenedione, 19-norandrostenedione and oestradiol-17β were obtained as major metabolites from androstenedione, while 19-norandrostenedione and oestradiol-17β were the major products from 19-hydroxyandrostenedione. Serum alone or serum plus FSH significantly enhanced formation of 19-norandrostenedione and oestradiol-17β from each substrate, compared with controls.

Micromolar concentrations (1 μmol/l) of 4-hydroxyandrostenedione, an aromatase inhibitor, significantly reduced formation of 19-norandrostenedione and oestradiol-17β by granulosa cells cultured with serum and FSH. Formation of 19-norandrostenedione and oestradiol-17β from androstenedione and 19-hydroxyandrostenedione was also significantly inhibited by aminoglutethimide phosphate, a cytochrome P-450 inhibitor known to block the conversion of androstenedione to oestrogens. Ketoconazole, an inhibitor of the cytochrome P-450 dependent 17,20-lysase, blocked formation of 19-norandrostenedione and oestradiol-17β only at millimolar concentrations. These results suggest that 19-norsteroid and oestrogen formation from C₁₉ aromatizable androgens may share a common or overlapping pathway, and imply that 19-norsteroid and oestrogen synthesis is mediated by cytochrome P-450 dependent enzymes.

Journal of Endocrinology (1989) 120, 251–260

ABSTRACT

The present studies were carried out to compare the low density lipoprotein (LDL) metabolism by freshly isolated immature porcine granulosa cells with that by luteal cells. Furthermore, we have examined the effect of serum used for plating of granulosa cells on lipoprotein degradation and utilization. In incubation studies, addition of LDL as an exogenous substrate had a mild stimulatory effect on progesterone accumulation by granulosa cells, while it exhibited a dose-dependent stimulatory effect on luteal cells. When granulosa and luteal cells were incubated with ¹²⁵I-labelled LDL, membrane binding of LDL occurred in both cell types, but only luteal cells were capable of internalizing the bound LDL. Granulosa cells in incubation degraded LDL much less in comparison with luteal cells, and the amount varied with the maturity of the cells. When granulosa cells were plated with graded amounts of serum which was withdrawn for 48 h following plating, they exhibited enhanced LDL degradation in a serum concentration-dependent fashion. Addition of serum for plating selectively enhanced utilization of LDL, but not high density lipoprotein (HDL) for progesterone accumulation by the cells in culture. Time-course studies on LDL degradation by granulosa cells following serum withdrawal indicate that the ability of cells to degrade LDL decreased in a time-dependent fashion. Serum withdrawal selectively decreased utilization of LDL but not HDL for progesterone secretion. It is concluded that immature granulosa cells have a limited capability to utilize cholesterol carried by LDL. However, when cultured in the presence of serum, cells acquire the ability to utilize more efficiently LDL-carried cholesterol for progesterone secretion which is then lost following long-term withdrawal of serum from culture.

Journal of Endocrinology (1989) 122, 557–564