Glutathione S-transferases (GSTs) are a family of soluble enzymes of detoxification that use reduced glutathione in conjugation and reduction reactions. Toxic electrophiles are substrates for the GSTs. GSTalpha is expressed at high levels in different tIssues such as the testis. Among the different GSTs present in the testis, GSTalpha is specifically expressed in Leydig and Sertoli cells known to be under the control of hormonal and local regulatory factors. The present study investigated the regulatory action of tumor necrosis factor-alpha (TNFalpha) on basal and hormone (FSH and testosterone)-stimulated GSTalpha expression in cultured Sertoli cells. Treatment with TNFalpha (0-20 ng/ml, 48 h) induced a decrease in basal GSTalpha mRNA levels in a dose-dependent manner (fivefold decrease; P<0.001). The maximal and half maximal effects were observed at 20 ng/ml and 7 ng/ml respectively. The inhibitory effect of TNFalpha was also time-dependent with a maximal inhibitory effect (threefold decrease; P<0.001) observed at 48 h. The inhibitory effect of the cytokine was also observed on basal GSTalpha protein (28 kDa) levels. TNFalpha also inhibited the hormone-stimulated GSTalpha expression in Sertoli cells. The treatment of cultured Sertoli cells with both FSH and TNFalpha (100 ng/ml and 10 ng/ml respectively, 48 h) resulted in a complete suppression of the stimulatory action of FSH on GSTalpha mRNA levels. Similarly, in Sertoli cells treated with testosterone or its non-aromatizable metabolite dihydrotestosterone (100 ng/ml, 24 h), TNFalpha reduced the hormone-stimulated GSTalpha mRNA and protein levels. TNFalpha inhibited basal GSTalpha expression without affecting mRNA stability. Indeed, the decay curves (mRNA half-life time=18 h) for the GSTalpha basal mRNA levels in Sertoli cells was similar in the absence or presence of TNFalpha (10 ng/ml, 48 h). Testosterone increased GSTalpha mRNA without affecting the enzyme mRNA stability. TNFalpha antagonized the androgen-stimulated GSTalpha mRNA levels without affecting the enzyme mRNA stability, suggesting that the interaction between the androgen and the cytokine is mostly exerted at a transcriptional level. FSH increased GSTalpha mRNA levels through an increase in mRNA stability (increased mRNA half-life times to 119 h). TNFalpha antagonized the stimulatory effect of FSH on GSTalpha mRNA levels by antagonizing the stabilizing effect exerted by the hormone on GSTalpha mRNA. Together, these results suggest that the increase in the cytokine levels within the testis would alter the detoxification processes against genotoxic products during spermatogenesis.

ABSTRACT

Thyroid cells cultured as monolayers on the porous bottom of culture chambers have been shown to express some specific functions of thyroid follicles. This system, which allows independent access to apical and basal media, is suitable for the long-term study of polarized processes, as the cells maintain their polarized organization. Iodination of thyroglobulin has been investigated under different culture conditions in the presence or absence of TSH. Apical thyroglobulin accumulation, apical iodide concentration and thyroglobulin iodination have been followed simultaneously. Iodide (0·5 μmol/l) was added to basal medium at various stages: only once for 4-day incubations and at each medium change or daily for longer experiments. TSH increased the amount of thyroglobulin secreted into the apical medium by five- to sixfold, whereas high basal iodide concentrations (> 5 μmol/l) inhibited thyroglobulin secretion by TSH-stimulated cells. TSH increased iodide uptake giving an iodide concentration ratio between apical and basal media of about 5. Thyroglobulin iodination was dependent upon TSH. Thyroglobulin was iodinated only in the apical compartment. Secretion and iodination of thyroglobulin were polarized phenomena, but the polarity of iodination was total whereas the polarity of secretion was only partial (10% basal secretion). This functional asymmetry was maintained for up to 29 days. The maximal incorporation of iodine into thyroglobulin obtained was never higher than 3·5 atoms/mol. Apical iodide concentrations from 1 to 15 μmol/l, depending on culture conditions, did not increase this value. These results suggest that cells cultured in this culture system are able to reproduce several steps of thyroidal iodide metabolism although there may be unknown factors which could interfere and reduce the efficiency of thyroglobulin iodination.

Journal of Endocrinology (1991) 128, 51–61

ABSTRACT

Individual large luteal cells (LLC) derived from pregnant swine differ conspicuously in their ability to secrete the peptide hormone relaxin under basal and stimulated conditions – the phenomenon of functional heterogeneity. The purpose of this study was to quantitate knowledge of this phenomenon through use of a reverse haemolytic plaque assay, a technique that utilizes antibody-directed, complement-mediated erythrocyte lysis to detect hormone secretion by single LLCs in culture.

Measurement of individual plaque areas (an index of the amount of relaxin secreted) demonstrated an approximate 100-fold range in the amount of relaxin secreted by a single cell under basal conditions. This range was doubled by exposure to the phorbol ester, 4β-phorbol 12β-myristate 13α-acetate (PMA; 50 nmol/l). Under basal conditions, 50 and 80% of the total amount of relaxin was secreted by approximately 10 and 30% of all LLCs respectively. The size of these fractions was not influenced by the time of incubation (1–8 h), or by the presence of either of two non-specific stimulatory relaxin secretagogues, PMA (50 nmol/l) or arachidonic acid (1 μmol/l). The unimodal frequency distribution of plaque areas (under basal or stimulated conditions) suggests that relaxin-secreting LLCs comprise a discrete functional population of secretory cells, at least under these experimental conditions.

We conclude that a remarkably small fraction of LLCs secretes the majority of relaxin, and that the size of this fraction was not influenced by time or secretagogues.

Journal of Endocrinology (1992) 134, 77–83

ABSTRACT

Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15 000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10–20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the α- and β-charge isomers of pheromaxein which were shown to have isoelectric points of 4·78 and 5·35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5α- and 5β-androstane series also showed some binding to pheromaxein, i.e. 17β-hydroxy-5α-androstan-3-one (19·2%), with 5α-androstan-3-one, which has a similar urinous odour to 5α-androst-16-en-3-one, showing the greatest binding (42·6%) relative to 5α-androst-16-en-3-one (100%).

J. Endocr. (1988) 118, 47–57

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

ABSTRACT

Veratridine, a sodium channel agonist, depolarized cultured thyroid cells and increased the secretion of radioiodine from the organically bound pool. These effects were similar to those of TSH. Depolarization of the cells by increasing the potassium concentration of the medium failed to promote secretion, indicating that the sodium influx, rather than the depolarization itself, mediated the response. Veratridine, like TSH, also acutely reduced the cells' iodide uptake and inhibited the iodide transport pump. Unlike TSH, however, veratridine reduced, rather than increased, the fractional exit rate of iodide anion from the unbound pool. The data are consistent with the hypothesis that a sodium influx mediates some but not all of the actions of TSH on the thyroid gland, including the stimulation of secretion of thyroid hormones.

J. Endocr. (1986) 110, 459–466

ABSTRACT

The time-course of changes in the nuclear 3,5,3′-tri-iodothyronine (T₃) receptor-binding capacity (B_max) of longissimus dorsi muscle has been investigated in cold-acclimated young pigs after a single large meal. Measurement of B_max values 4, 8, 12 and 24 h after feeding indicated a decline in receptor numbers after food intake with the lowest values occurring at 8 h. The receptor numbers then increased significantly, with the values at 12 h being more than 50% higher than those obtained at 8 h. The B_max values reached their highest level 24 h after feeding. No significant changes in the dissociation constant were observed. Possible reasons for the changes in T₃ receptor numbers are discussed and it is suggested that the increase in T₃ receptor numbers 12–24 h after feeding may play a role in enhancing the thermogenic capacity of the tissues in response to food.

Journal of Endocrinology (1992) 134, 67–72

Abstract

Thyroid hormones have been implicated in the regulation of nuclear 3,5,3′-tri-iodothyronine (T₃) receptor binding capacity (B_max) but, despite numerous in vivo and in vitro studies, there is considerable controversy regarding their exact role. Since changes in thyroid status alter energy balance and hence may influence T₃ receptor numbers, the effects of chronic hypothyroidism and T₄ treatment have been studied in young pigs under conditions of controlled energy intake. Four groups of animals comprising a hypothyroid, a euthyroid and a hyperthyroid group, all on the same level of food intake, and a hyperthyroid group on twice the amount of food were used. After 3 weeks on the treatment regimes, both the hypothyroid animals on the same level of food intake and the hyperthyroid animals on twice the amount of food had significandy increased B_max values (97% and 137% higher respectively) compared with euthyroid controls. However, there was no difference between controls and the hyperthyroid animals on the same level of food intake. In a second study, the effects of short-term treatment of euthyroid animals with T₃ was investigated. Results showed that in two groups of controls that received intravenous saline, those on a higher food intake had higher B_max values (76% increase). Intravenous T₃ administration to animals on a low food intake did not change the receptor numbers. In none of the studies was there any change in the dissociation constant of the receptors as a result of different treatments. It is suggested that, at least in postnatal life, thyroid hormones per se have no significant effect on nuclear T₃ receptor numbers in skeletal muscle. Instead, changes in B_max in response to thyroid status may be secondary to alterations in energy balance induced by these hormones.

Journal of Endocrinology (1995) 144, 233–242

ABSTRACT

The effects of insulin-like growth factor-I (IGF-I) on aromatase P450 activity and steroid production in preimplantation pig conceptuses were evaluated in vitro. Conceptuses recovered from gilts on days 10 and 12 of pregnancy were incubated for 6 h in modified Eagle's Minimum Essential Medium (MEM) plus IGF-I (0·1 μg/ml) or insulin (8·5 μg/ml), and conceptuses were monitored for their ability to convert [1,2-³H]β-testosterone into oestrogens. Aromatase activity of day-10 conceptuses was low and unaffected by IGF-I or insulin. In contrast, basal aromatase activity in day-12 conceptuses was about threefold higher and was further increased by IGF-I (P < 0·02), but was unaffected by insulin. To determine whether higher aromatase P450 activity was associated with increased oestradiol production, concentrations of oestradiol were determined by radioimmunoassay in culture medium of day-11 and -12 conceptuses, after incubation in MEM alone or in the presence of dehydroepiandrosterone (DHA, 1 μg/ml) with or without IGF-I (0·1 μg/ml) or insulin (0·1 or 8·5 μg/ml) for 24 h. Conceptuses in MEM plus DHA produced more oestradiol (P < 0·01) than those in MEM alone. Addition of IGF-I or insulin did not increase the effect of DHA. Basal oestradiol production was dependent on conceptus size; however, IGF-I or insulin did not affect basal or DHA-stimulated oestradiol production regardless of conceptus size. These findings demonstrate that IGF-I can modulate aromatase activity in vitro, without affecting overall de-novo steroidogenesis. Thus, the developmental increase in conceptus oestradiol production observed during early pregnancy in the pig may reflect synergistic interactions between IGF-I and other regulatory factors present within the conceptus and/or uterine environment.

Journal of Endocrinology (1991) 130, 245–250

The role of human chorionic somato-mammotrophin (HCS), a postulated 'growth hormone of pregnancy', on the transport of nutrients through the placenta needs to be investigated. As a simpler alternative we have studied the effects of HCS on the uptake of [¹⁴C]α-aminoisobutyric acid ([¹⁴C]AIB) by the immature mouse uterus and ovary. The stimulatory effect of growth hormone (GH) on [¹⁴C]AIB uptake by the combined uterus and ovary of the rat has been shown previously (Noal, Riggs, Walker & Christensen, 1957). Mice used were of CSI strain and were 21 to 23 days old. Human chorionic somato-mammotrophin (Nutritional Biochemicals) and GH (Sigma Chemical Co. Ltd) were injected intraperitoneally in 0·1 ml 0·15m-NaCl. Control mice received saline alone. Methods previously described (Rosenberg, Blair & Segal, 1961; Rosenberg, Downing & Segal, 1962) were adapted for estimation of total water content of the tissue, determination of extracellular fluid (ECF) with