bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin D hydroxylases and their regulation in a naturally vitamin D-deficient subterranean mammal, the naked mole rat (Heterocephalus glaber)
Authors: Buffenstein R. , Sergeev I. N. , Pettifor J. M.
Published: December 2008

The vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is generated by a series of hydroxylation steps in the liver and kidneys. We investigated whether naturally vitamin D-deficient subterranean mammals (naked mole rats, Heterocephalus glaber) employ the same enzymatic pathways, and whether these are regulated in a similar manner to that established for other mammals.

Vitamin D3-25-hydroxylase in the liver and both 25-hydroxyvitamin D3-l-hydroxylase and 25-hydroxyvitamin D3-24 hydroxylase (1-OHase and 24-OHase) in the kidney were detectable in mole rats. As expected for vitamin D-deficient mammals, the 1-OHase activity predominated over the 24-OHase. After mole rats received a supraphysiological supplement of vitamin D3, 1-OHase activity was suppressed and 24-OHase activity was enhanced. Irrespective of vitamin D status, forskolin (a protein kinase A activator) and dibutyryl cyclic AMP did not alter the activity of either 1-OHase or 24-OHase. These findings suggest that the response of renal hydroxylases to parathyroid hormone was blunted. Phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) (protein kinase C activators), suppressed 1-OHase activity. 24-OHase activity was induced by TPA but not by OAG. These effects were similar to those illicited by vitamin D3 supplementation but were additive in that they increased the responses shown in vitamin D-replete mole rats.

These data confirm that naturally vitamin D-deficient mole rats can convert vitamin D3 to the hormone, 1,25(OH)2D3. Furthermore, the enzymes 1-OHase and 24-OHase present in the kidneys of these mammals are regulated independently by 1,25(OH)2D3 and protein kinase C-mediated pathways of intracellular signalling, but are not regulated by the cyclic AMP–protein kinase A signal transduction pathway.

Journal of Endocrinology (1993) 138, 59–64
Phospholipid methylation decreases in human chorionic gonadotrophin-induced desensitized rat Leydig cells
Authors: Ronco A. M. , Valladares L. E.
Published: December 2008

Phospholipid methylation in Leydig cells from desensitized rats was studied. The incorporation of l-[methyl-3H] methionine into phospholipids in intact Leydig cells decreased when animals were injected with a single high dose of human chorionic gonadotrophin (hCG). This effect was detected on the first day after hCG injection and remained up to 12 days after treatment. The inhibition was not due to a reduced uptake of l-[methyl-3H]methionine. A decreased phospholipid methylation with unaltered phospholipid methyltransferase activity was observed on days 1, 6 and 12 after the hCG. On day 3 after hCG injection, phospholipid methyltransferase activity and phospholipid methylation in intact Leydig cells were both inhibited by 40%. Also, a minimal amount of LH free receptors and the lowest number of total receptors was observed at this time. Thus, a relationship between the reduced enzymatic activity and the maximal decrease in LH surface receptors is suggested. In addition, the decreased incorporation of l-[methyl-3H]methionine into phospholipids on days 1, 6 and 12 after hCG injection, could be associated with other cellular changes related to the desensitization process.

Journal of Endocrinology (1993) 138, 65–71
Time-course of vertebral and femoral bone loss in rats administered gonadotrophin-releasing hormone agonist
Authors: Kurabayashi T. , Fujimaki T. , Yasuda M. , Yamamoto Y. , Tanaka K.
Published: December 2008

This study was carried out (1) to compare the time-course of the change in bone metabolism in rats administered gonadotrophin-releasing hormone agonist (GnRHa) and bilaterally ovariectomized (OVX) rats and (2) to investigate the changes in bone metabolism after discontinuance of GnRHa.

Seventy female Sprague–Dawley rats, aged 90 days, were divided into four groups. Group 1 underwent a sham operation, group 2 was surgically ovariectomized and group 3 was given a GnRHa (leuprorelin acetate for depot suspension) s.c. injection every 30 days. Group 3 was further divided into three subgroups: rats were administered GnRHa for 12 months (GnRHa 12M), 6 months (GnRHa 6M) or 3 months (GnRHa 3M). Group 4 served as a basal control. The bone mineral density (BMD) of lumbar vertebrae and femoral bone, measured by dual-energy X-ray absorptiometry, and the serum bone metabolic parameters were determined every 45–90 days. The bone histomorphometry of lumbar vertebra was measured on days 180 and 360 after surgery.

GnRHa 12M rats showed significantly lower BMD of vertebrae and femoral bone, lower bone volume and higher bone turnover compared with sham-operated rats and those with secondary hyperparathyroidism on days 180 and 360. Their time-course for changes in bone metabolism was almost the same as that of OVX rats. GnRHa-discontinued rats showed a recovery of bone turnover. The recovery of BMD in GnRHa 6M rats was smaller than that of GnRHa 3M rats after GnRHa discontinuance. The bone volume for GnRHa 6M rats was significantly lower than that for GnRHa 3M on day 360.

Journal of Endocrinology (1993) 138, 115–125
Influence of endogenous cholinergic tone and α-adrenergic pathways on growth hormone responses to His-d-Trp-Ala-Trp-d-Phe-Lys-NH2 in the dog
Authors: Muruais J. , Peñalva A. , Dieguez C. , Casanueva F. F.
Published: December 2008

His-d-Trp-Ala-Trp-d-Phe-Lys-NH2 (GHRP-6) is a synthetic peptide unrelated to any known hypothalamic-releasing hormone including growth hormone-releasing hormone (GHRH). Interestingly, this peptide induces a dose-related increase in plasma GH levels in all species tested so far. The aim of this study was to investigate the action of GHRP-6 alone or in combination with GHRH on GH release in dogs. In addition, the activation or blockade of endogenous cholinergic tone and α-1 adrenoceptors on GHRP-6-stimulated GH secretion was assessed.

In adult Beagle dogs (n = 10), GHRP-6 (90 μg i.v.) increased basal GH levels from 2·6 ± 1·5 to 14·4 ± 3·1 μg/l (mean ± s.e.m.) after 15 min. GHRH (50 μg i.v.) induced a GH peak of 9·7 ± 2·2 μg/l at 15 min. The combined administration of GHRP-6 and GHRH strikingly potentiated canine GH release with a peak of 54 ± 9·0 μg/l (P <0·01). Pretreatment with the cholinergic agonist pyridostigmine (30 mg per os) increased GHRP-6-stimulated GH secretion (37·9 ± 10·1 μg/l P <0·05), while the muscarinic blocker atropine (100 μg i.v.) completely abolished (GH peak lower than 2 μg/l) the stimulatory action of GHRP-6. On the other hand, administration of the α-2 adrenergic agonist clonidine (4 pg/kg i.v.) increased basal plasma GH levels without affecting GH responses to GHRP-6. Finally, while the α-1 adrenergic agonist methoxamine (5 mg i.v.) did not significantly increase GH responses to GHRP-6, administration of the α-1 adrenoceptor antagonist prazosin (20 mg i.v.) reduced GHRP-6-induced GH secretion (area under curve, 206 ± 39 vs 557 ± 172, P <0·05).

In summary, the synergistic effect of the combined administration of maximal doses of GHRP-6 and GHRH suggests that these two peptides act through different mechanisms. The finding that cholinergic drugs were able to modulate the GH secretion elicited by GHRP-6 argues against the hypothesis that such a peptide acts by influencing hypothalamic somatostatin release and suggests that it acts directly at the pituitary level. Finally, the unexpected lack of effect of clonidine and the inhibitory effect of prazosin on GHRP-6-induced GH secretion suggests that the role of α-adrenergic pathways in GH secretion is more complex than previously thought.

Journal of Endocrinology (1993) 138, 211–218
Changes induced by cadmium administration on thyroxine deiodination and sulfhydryl groups in rat liver
Authors: Paier B. , Hagmüller K. , Noli M. I. , Pondal M. Gonzalez , Stiegler C. , Zaninovich A. A.
Published: December 2008

The effects of cadmium on 5′-deiodination of thyroxine (T4) by rat liver and on the hepatic concentration of non-protein sulfhydryl groups (NPSH) were studied in Wistar rats of 200–250 g body weight. A group of ten rats was injected with cadmium chloride (300 μg/100 g body weight i.p.) daily for 4 days. Another group of six rats received, in addition, dithiothreitol (DTT; 1 mg/100 g body weight i.p.) daily for the same period. A group of eight normal untreated rats served as control. T4 deiodination was also determined in aliquots of liver from untreated rats, with cadmium (2 or 5 mmol/l) and with or without DTT (0, 2·5, 5 or 10 mmol/l) plus 1 μCi 125I-labelled T4. Hepatic NPSH were measured by a colorimetric method employing dithioldinitrobenzoic acid. Homogenates were incubated for 90 min at 37 °C and chromatographed in a tertiary amyl alcohol: hexane: ammonia (2 mol/l) (10: 1: 12) system. Cadmium-injected rats showed a significant (P <0·01) decrease in T4 deiodination and in the generation of 125I (P <0·01) and tri-iodothyronine (T3) (P <0·02). NPSH were also decreased (P <0·02). Administration of DTT restored T4 deiodination and NPSH to normal. In-vitro addition of cadmium or DTT to normal rat liver homogenates induced similar effects on the degradation of T4. Serum concentrations of T4 (P <0·01) and T3 (P <0·01) declined significantly in cadmium-injected rats, whereas DTT administration failed to normalize serum hormone levels. The data suggest that cadmium may have decreased 5′-deiodinating activity through binding to sulfhydryl groups of 5′-deiodinase as it does in other enzymes. The effects on serum T4 concentrations may be unrelated to those on 5′-deiodinase.

Journal of Endocrinology (1993) 138, 219–224
Growth hormone receptor regulation in growth hormone-deficient dwarf rats
Authors: Carmignac D. F. , Robinson I. C. A. F. , Enberg B. , Norstedt G.
Published: December 2008

In the rat, many actions of GH depend upon the sexually dimorphic pattern of exposure to GH. Hepatic human GH (hGH) receptor binding differs between the sexes and is sensitive to GH deficiency, but this has mostly been studied in acutely hypophysectomized rats, which lack all pituitary hormones. We have used a strain of GH-deficient dwarf (Dw) rats to determine whether chronic GH deficiency alters the normal developmental pattern and sexually dimorphic expression of hepatic GH receptors. Adult female Dw rats had lower levels of 125I-labelled hGH binding (reflecting predominantly lactogenic receptors) than their normal counterparts whereas there was no difference between adult Dw and normal males; binding capacity increased from 25 days of age, becoming sexually dimorphic from 40 days to adulthood in both strains (% specific binding/mg protein: normal males 1·6 ± 0·3, normal females 13·2 ± 1·1, Dw males 2·1 ±0·4, Dw females 10·0 ± 0·6). In contrast, hepatic 125I-labelled bovine GH (bGH) binding (somatogenic receptors) was much lower, and similar in both Dw and normal animals. A sex difference in 125I-labelled bGH binding was only seen in adult animals, and was considerably less marked in Dw rats compared with normal animals (normal males 1·3 ±0·1, normal females 2·5 ± 0·2, Dw males 1·9 ± 0·2, Dw females 2·4 ± 0·2%/mg protein). Continuous hGH infusion stimulated growth in female Dw rats, and raised somatogenic and lactogenic GH binding (3·2 ± 0·4 and 19·6 ± 2·5%/mg protein) compared with sham-infused controls (2·4 ± 0·2 and 7·9 ± 0·6%/mg protein). Dw rats had significantly smaller amounts of hepatic GH receptor mRNA than normal rats, but there was no significant sex difference in GH receptor mRNA levels in the dwarfs. The pituitary GH deficiency in Dw rats was present at birth and the relative deficit remained constant despite large increases in pituitary GH that occurred from birth to maturity. Thus whilst hepatic GH receptor expression can be altered by GH in Dw rats, their chronic GH deficiency causing severe dwarfism is accompanied by only small differences in hepatic GH receptor expression.

Journal of Endocrinology (1993) 138, 267–274
Expression of mRNA coding for pituitary hormones and pituitary-specific transcription factor in the pituitary gland of the rdw rat with hereditary dwarfism
Authors: Shibayama K. , Ohyama Y. , Ono M. , Furudate S.
Published: December 2008

The rdw rat (gene symbol: rdw) with hereditary dwarfism has been shown immunohistochemically to have subnormal numbers not only of GH- but also of prolactin- and thyrotrophin-positive cells. To characterize the dwarfism of this strain, the expression of pituitary hormone mRNAs was examined by Northern hybridization. The pituitary gland in the rdw rat expressed 30–100 times less GH and prolactin mRNAs than normal controls, whereas mRNAs for pro-opiomelanocortin and the α subunit of rat glycoprotein hormone revealed a significant increase. There was a non-significant difference in rat LH-β subunit and FSH-β subunit between normal and rdw rats. The suppressed expression of a pituitary-specific transcription factor, Pit-1, is considered to cause hereditary dwarfism in mouse strains Snell and Jackson, whose phenotypes resemble those of the rdw rat. In this study, however, no difference in mRNA expression for Pit-1 was found between rdw rats and controls. This work indicates that the rdw rat may not have the same genotype as the phenotypically similar dwarf mice, Snell, Jackson and Ames.

Journal of Endocrinology (1993) 138, 307–313
Variability in the urinary excretion of growth hormone in children: a comparison with other urinary proteins
Authors: Skinner A. M. , Clayton P. E. , Price D. A. , Addison G. M. , Mui C. Y. W.
Published: December 2008

As a basis for assessment of the clinical validity of urinary GH (uGH) measurements in children, the day-to-day variability in renal handling of GH has been compared with that of albumin, N-acetylglucosaminidase (NAG) and creatinine. Five overnight urine specimens were collected over a 2-week period from 78 healthy children (aged 5–16 years), 20 of normal stature and 58 with growth disorders; ten children were classified as GH-deficient (GHD) and 48 were designated short normal (SN).

The variability of excretion of each substance was expressed as a coefficient of variation (C.V.) which was not influenced by expressing the urine results as total mass excreted, concentration, excretion rate or as a ratio to creatinine. There was considerable night-to-night variability in the excretion of all substances (mean C.V. values for all groups: 56% for albumin, 41% for GH, 33% for NAG and 27% for creatinine). No differences were found in the variability of GH excretion between males and females, nor between prepubertal and pubertal subjects. The mean C.V. for uGH excretion ranged from 37% in normal and 35% in SN children to 52% in those with GHD (P <0·05). Assay variation rather than a change in renal protein handling accounted for the large variations in uGH concentrations of <5 pg/ml, thus contributing to the high uGH C.V. of the GHD group. Increasing the number of samples collected (up to five) decreased the expected sample variation (error) for uGH but not significantly and only improved efficiency in the diagnosis of growth disorders from 91 to 95%, while reducing the convenience and practicality of the test.

These results indicate that variation in urinary protein excretion over a 2-week period is considerable (albumin>GH>NAG) in both normal children and those with growth disorders. To apply this test to routine clinical management, we recommend comparison of a single overnight uGH measurement with normal ranges derived from age-, sex- and pubertal status-matched children.

Journal of Endocrinology (1993) 138, 337–343
Plasma somatolactin concentrations in salmonid fish are elevated by stress
Authors: Rand-Weaver M. , Pottinger T. G. , Sumpter J. P.
Published: December 2008

The preliminary finding that plasma levels of somatolactin (SL) were markedly elevated following stress caused by confinement in chinook salmon (Oncorhynchus tshawytscha) prompted a more detailed study of SL dynamics during stress. SL levels have been determined in the plasma of rainbow trout (Oncorhynchus mykiss) during exposure to acute (0–30 min) and short (0–24 h) periods of stress resulting from handling and confinement. The results show that SL levels increase rapidly within minutes following the onset of stress, reach a peak between 1 and 2 h, decline over the next 3 h, and then show an additional increase again by 24 h. During acute stress caused by confinement, the increase in plasma SL levels occurred within 2 min, thus showing a more rapid response than cortisol. This suggests that the response is mediated directly by the hypothalamus and is not a result of a feedback mechanism. The results also demonstrate that SL secretion in response to stress is at least partially under genetic control. In the short-term stress experiment, progeny of fish selected as high responders or low responders to stress, based on the magnitude of the plasma cortisol levels induced by stress, were used, and these fish showed similarly accentuated or attenuated release of SL following stress. These results clearly demonstrate that non-specific environmental stress causes rapid activation of SL-secreting cells in the pars intermedia, suggesting that this hormone has an important role in the adaptive response of fish to stress.

Journal of Endocrinology (1993) 138, 509–515
Regulation of 11β-hydroxysteroid dehydrogenase by sex steroids in vivo: further evidence for the existence of a second dehydrogenase in rat kidney
Authors: Low S. C. , Assaad S. N. , Rajan V. , Chapman K. E. , Edwards C. R. W. , Seckl J. R.
Published: December 2008

11β-Hydroxysteroid dehydrogenase (11β-OHSD) catalyses the reversible conversion of corticosterone to inactive 11-dehydrocorticosterone, thus regulating glucocorticoid access to mineralocorticoid and perhaps glucocorticoid receptors in vivo. 11β-OHSD has been purified from rat liver and an encoding cDNA isolated from a liver library. However, several lines of indirect evidence suggest the existence of at least two isoforms of 11β-OHSD, one found predominantly in glucocorticoid receptor-rich tissues and the other restricted to aldosterone-selective mineralocorticoid target tissues and placenta. Here we have examined the effects of chronic (10 day) manipulations of sex-steroid levels on 11β-OHSD enzyme activity and mRNA expression in liver, kidney and hippocampus and present further evidence for the existence of a second 11β-OHSD isoform in kidney.

Gonadectomized male and female rats were given testosterone, oestradiol or blank silicone elastomer capsules, controls were sham-operated. In male liver, gonadectomy+ oestradiol treatment led to a dramatic decrease in both 11β-OHSD activity (69 ± 8% decrease) and mRNA expression (97 ± 1% decrease). Gonadectomy and testosterone replacement had no effect on male liver 11β-OHSD. However, in female liver, where 11β-OHSD activity is approximately 50% of that in male liver, gonadectomy resulted in a marked increase in 11β-OHSD activity (120 ± 37% rise), which was reversed by oestradiol replacement but not testosterone treatment.

In male kidney, gonadectomy+oestradiol treatment resulted in a marked increase in 11β-OHSD activity (103 ± 4% rise). By contrast, 11β-OHSD mRNA expression was almost completely repressed (99 ± 0·1% decrease) by oestradiol treatment. This effect of oestradiol was reflected in a loss of 11β-OHSD mRNA in all regions of the kidney showing high expression by in-situ hybridization. In female kidney, oestradiol replacement also led to an increase in 11β-OHSD activity (70 ± 15% rise) while mRNA expression fell by 95 ± 3%. None of the treatments had any effect on enzyme activity or mRNA expression in the hippocampus, although transcription starts from the same promoter as liver.

We conclude that (i) sex steroids regulate 11β-OHSD enzyme activity and mRNA expression in a tissue-specific manner and (ii) the concurrence of increased enzyme activity with near absent 11β-OHSD mRNA expression in the kidney following oestradiol treatment suggests that an additional gene product is responsible, at least in part, for the high renal activity observed.

Journal of Endocrinology (1993) 139, 27–35
Regulation of serum insulin-like growth factor-I (IGF-I), hepatic growth hormone binding and IGF-I gene expression in the rat during pregnancy and lactation
Authors: Travers M. T. , Madon R. J. , Flint D. J.
Published: December 2008

An apparent GH resistance occurs in pregnancy, since GH concentrations in serum are reported to be normal or elevated, whereas serum IGF-I falls to very low levels. To determine whether this GH resistance is manifest at the level of the hepatic GH receptor or in the ability of GH to initiate IGF-I gene expression, we have determined hepatic IGF-I mRNA expression, circulating IGF-I and hepatic GH binding during various stages of pregnancy and lactation in the rat. The concentration of IGF-I in serum fell from 37 ± 5 nmol/l (means ± s.e.m.) in virgin rats to 17 ± 1 nmol/l in rats in late pregnancy, recovered in early lactation (31 ± 3 nmol/l) but was again significantly lower than in virgin animals by mid-lactation (22 ± 3 nmol/l).

Hepatic GH binding did not vary significantly during pregnancy but showed a small significant decrease in early lactation when expressed per mg membrane protein. When expressed as GH binding per liver, however, there were no significant changes in GH binding at any stage. Liver weight increased significantly between virgin and early pregnant animals (7·1 ± 0·2 g compared with 9·2 ± 0·5 g respectively, P<0·01) and continued to increase up to late lactation (14·3 ± 0·4 g).

Similarly, although the amount of IGF-I gene expression/unit RNA declined in late pregnant when compared with virgin animals (6·0 ± 0·6 versus 3·4 ± 0·4 arbitrary optical density units respectively, P<0·05), when the increase in liver weight and RNA content during pregnancy was taken into account there was actually a steady increase in IGF-I gene expression per total liver throughout both pregnancy and lactation, the difference becoming significant by mid-lactation (P<0·05). Thus, when the changes in liver weight which take place during pregnancy and lactation are taken into account we conclude that any hepatic resistance to GH in late pregnancy and lactation must be a post-receptor event which is mitigated in large part by increase in liver size, cell number and RNA content.

Journal of Endocrinology (1993) 139, 89–95
Suppressed expression of the cytochrome P45017α protein in the testicular feminized (Tfm) mouse testes
Authors: Le Goascogne C. , Sananès N. , Gouézou M. , Baulieu E. E. , Robel P.
Published: December 2008

The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P45017α activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabelled in the testes of normal and Tfm adult (2·5–6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17α-hydroxylase,C17→20 lyase (P45017α) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 °C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 μmol/l). All Leydig cells remained positive for P450scc and 3β-HSD, and P45017α became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P45017α protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon.

Journal of Endocrinology (1993) 139, 127–130
Investigation of mechanisms mediating the increase in plasma concentrations of thyroid hormones after a meal in young growing pigs
Authors: Dauncey M. J. , Morovat A.
Published: December 2008

These studies investigated a number of possible mechanisms which could mediate the increase in plasma concentrations of thyroid hormones after a meal in young growing pigs. It has been established that in animals fed one meal a day, an immediate rise in plasma 3,5,3′-tri-iodothyronine (T3) and a slightly delayed increase in thyroxine (T4) levels are followed by a more sustained peak in both hormones several hours later. The increase in thyroid hormones involves both total and free T3 and T4, and there is no change in plasma albumin, the high-capacity thyroid hormone-binding protein in the pig. It has also been shown that the immediate rise in plasma T3 is not mediated either by an increase in plasma glucose concentration or by neural mechanisms associated with distension of the gastrointestinal tract. However, the finding that plasma T3 increases rapidly after feeding in thyroidectomized animals maintained on a replacement dose of T4 alone, indicates the source of T3 to be non-thyroidal.

It is concluded that the rise in plasma thyroid hormones after a meal depends on the energy content of the food but not directly on the circulating glucose levels. The immediate increases in plasma T3 and T4 are probably due largely to a redistribution of the hormonal pools, and peripheral 5′-monodeiodination of T4 may also contribute significantly to the post-prandial rise in T3. The potential significance of these findings in relation to both the metabolic and growth-promoting effects of thyroid hormones is discussed.

Journal of Endocrinology (1993) 139, 131–141
Ovarian and hepatic insulin-like growth factor-I gene expression and associated metabolic responses in prepubertal gilts subjected to feed restriction and refeeding
Authors: Charlton S. T. , Cosgrove J. R. , Glimm D. R. , Foxcroft G. R.
Published: December 2008

The effects of feed restriction and refeeding on ovarian and hepatic insulin-like growth factor-I (IGF-I) gene expression, systemic and ovarian IGF-I concentrations and on associated metabolic changes were measured in prepubertal gilts. Eleven pairs of littermate gilts (70·7 ± 4·7 kg) were placed on a maintenance level of feeding for 7 days (days 1–7). On day 8, littermates were either fed at a maintenance level of energy or fed to appetite for a further 6 days. Blood samples were taken on day 13 (07.00–16.00 h) to determine plasma insulin and IGF-I, and on day 14 (02.00–06.00 h) to determine plasma GH levels. Following slaughter on day 14, one ovary from each animal was retained to measure follicular fluid IGF-I and oestradiol concentrations. The remaining ovary and a sample of liver were retained for IGF-I mRNA analysis using a ribonuclease protection assay. Six days of refeeding significantly increased plasma IGF-I (P<0·005) and basal insulin (P<0·05) but there was no effect on plasma GH. Ovarian follicular volume and diameter were significantly larger after refeeding (P<0·05), with no effect on follicular fluid oestradiol concentrations. Mean follicular fluid IGF-I concentrations were unaffected by treatment. However, the relationships between individual follicular IGF-I concentrations, absolute follicular fluid IGF-I contents and follicle volume were affected by feeding level (P<0·05). Regression analysis of the same data also revealed that at this stage of maturity, small follicles had greater follicular fluid concentrations of IGF-I than larger follicles. Refeeding increased the amount of IGF-I mRNA in hepatic but not ovarian tissue. We conclude that there is differential regulation of the IGF-I gene in porcine hepatic and ovarian tissues, and that ovarian factors other than, or as well as, IGF-I are involved in the regulation of ovarian responses to refeeding.

Journal of Endocrinology (1993) 139, 143–152
The influence of the maternal thyroid hormone environment during pregnancy on the ontogenesis of brain and placental ornithine decarboxylase activity in the rat
Authors: Pickard M. R. , Sinha A. K. , Ogilvie L. , Ekins R. P.
Published: December 2008

The influence of maternal hypothyroxinaemia on early brain and placental development was examined in a partially thyroidectomized (parathyroid-spared; TX) rat dam model. Ornithine decarboxylase (ODC) specific activity, along with more general indices of cell growth, were determined in prenatal whole brain (at 15, 19 and 22 days of gestation), postnatal brain regions (at 5, 10 and 14 days) and placenta.

Maternal hypothyroxinaemia resulted in reductions in fetal body weight, brain weight, brain DNA content and brain total protein content at 15 days of gestation; the latter effect persisting until 19 days of gestation. Further changes in brain cell growth were observed near term, when an increase in the DNA concentration was accompanied by a decrease in the total protein: DNA ratio. Growth of the postnatal brain regions appeared normal, with the exception of an isolated increase in the protein content of the cerebellum at postnatal day 5. Determination of the specific activity of brain ODC revealed a complex pattern of change in the progeny of TX dams, super-imposed upon the normal ontogenetic decline. In the fetal brain, activity was initially deficient at 15 days of gestation but was increased at 22 days of gestation relative to controls. The compromise extended into the postnatal period; ODC specific activity being transiently reduced in the brainstem, the subcortex and the cerebral cortex. Placental development was less consistently affected; wet weight, gross indices of cell growth (DNA content, DNA concentration, total protein: DNA ratio) and ODC specific activity were all normal in the TX dam. However, cytosolic and total protein concentrations were reduced at 15 and 19 days of gestation respectively.

These results demonstrate abnormal fetal brain cell development as a consequence of maternal hypothyroxinaemia. The damage extended into the neonatal period, well after the onset of fetal thyroid hormone synthesis. Although the reduced supply of maternal thyroxine to the fetal brain may play a major role in this dysgenesis, factors such as the impairment of placental function must be taken into consideration.

Journal of Endocrinology (1993) 139, 205–212
Adrenal progesterone facilitates the negative feedback of oestrogen on LH release in ovariectomized rats
Authors: Salicioni A. M. , Carón R. W. , Deis R. P.
Published: December 2008

There is evidence that the adrenals play a role in the regulation of the synthesis and release of gonadotrophins in various vertebrates. The aim of this study was to determine the part played by adrenal steroids, with special reference to progesterone, on the concentration of LH in ovariectomized (OVX) and oestrogen-primed rats. OVX rats received a single s.c. injection of vehicle or oestradiol benzoate (OB, 20 μg/rat). This day was designated as day 0. Three or four days later (day 3–day 4), the rats were treated with mifepristone (10 mg/kg) or with two doses of progesterone antiserum and blood samples were obtained at 13.00 and 18.00 h. OB treatment of OVX rats reduced serum LH at 13.00 h and 18.00 h on day 3 but only at 13.00 h on day 4. The administration of mifepristone at 08.00 h to OVX and oestrogen-treated rats induced a significant increase in serum LH at 18.00 h on days 3 and 4, without modifying the values at 13.00 h. When mifepristone was given at 13.00 h a much larger increase in serum LH was obtained at 18.00 h. In OVX and oestrogen-treated rats, adrenalectomy on day 2 (08.00–09.00 h) induced an increase in serum LH at 18.00 h similar to that observed in the OVX and oestrogen-primed rats after mifepristone treatment. In order to determine the specificity of the effect of mifepristone, a group of OVX and oestrogentreated rats was injected with progesterone antiserum at 08.00 and 13.00 h on day 3. Serum LH concentrations at 13.00 and 18.00 h on day 3 were similar to values obtained in OVX rats treated with oestrogen and mifepristone. Serum progesterone was measured at 08.00 and 13.00 h in OVX and OVX and oestrogenprimed rats. At both times, values were similar in OVX rats but oestrogen treatment significantly increased serum progesterone levels. The important role of adrenal progesterone on the regulation of LH secretion in OVX and oestrogen-primed rats is evident from these results. Blocking progesterone action at the receptor level, we showed that OB significantly increased LH values at 18.00 h. On the basis of these studies it is tempting to speculate on the possibility of an inhibitory or stimulatory effect of oestrogen on serum LH concentration in OVX rats, according to the presence or absence of adrenal progesterone action.

Journal of Endocrinology (1993) 139, 253–258
Effects of insulin-like growth factor-I and its analogues on bovine hydrogen peroxide release by neutrophils and blastogenesis by mononuclear cells
Authors: Zhao X. , McBride B. W. , Trouten-Radford L. M. , Burton J. H.
Published: December 2008

The biological potencies of recombinant human insulin-like growth factor-I (IGF-I) and two of its analogues were examined for hydrogen peroxide release by neutrophils and blastogenesis by mononuclear cells. The binding affinities of these peptides for bovine serum IGF-binding proteins (IGFBPs) and IGF-I receptors on bovine neutrophils and mononuclear cells were also investigated. Relative to control treatment containing no IGF-I, preincubation of neutrophils with 12·5 μg/l of IGF-I, des(1–3)IGF-I (an analogue of human IGF-I lacking the N-terminal tripeptide Gly-Pro-Glu) and long R3 IGF-I (an analogue of human IGF-I with arginine replacing glutamate at position 3 of human IGF-I and the N-terminal extension Met-Phe-Pro-Ala-Met-ProLeu-Ser-Ser-Leu-Phe-Val-Asn) increased the release of H2O2 by 65%, 64% and 32% respectively. However, the difference in stimulating the release of H2O2 between long R3 IGF-I and other two (IGF-I and des(1–3)IGF-I) was reduced at a dosage of 100 μg/l. In the absence or presence of 2·5% fetal calf serum (FCS), 100 μg/l of IGF-I, des(1–3)IGF-I but not long R3 IGF-I significantly stimulated thymidine incorporation into mononuclear cells. In addition, des(1–3)IGF-I was more potent than IGF-I in stimulating thymidine incorporation into mononuclear cells in the presence of 2·5% FCS. IGF-I displaced 125I-labelled IGF-I binding to serum IGFBPs with half-maximal inhibitory concentrations of approximately 1·5 nmol/1, while des(1–3)IGF-I and long R3 IGF-I only inhibited binding by 20% and 6% respectively, even at a concentration of 35 nmol/l. Similar affinities for IGF-I receptors on neutrophils and mononuclear cells were shown for IGF-I and des(1–3)IGF-I. Conversely, much lower affinities for these receptors were demonstrated for long R3 IGF-I. These results suggest that the biological activities of IGF-I and its analogues in cells of the bovine immune system depend on their binding characteristics both to receptors and to binding proteins.

Journal of Endocrinology (1993) 139, 259–265
11β-Hydroxylase gene expression in the rat adrenal cortex
Authors: Ho M. M. , Vinson G. P.
Published: December 2008

It is now known that in the rat there are two distinct species of cytochrome P45011β/18, namely aldosterone synthase and 11β-hydroxylase. Whereas aldosterone synthase is located exclusively in the zona glomerulosa, the zonal distribution and site of production of 11 β-hydroxylase is not entirely clear. In the present study we examined the zonal expression of 11β-hydroxylase mRNA in adrenals from control rats and animals subjected to ACTH treatment and dietary sodium restriction using a non-isotopic in-situ hybridization technique. The results were compared with those obtained using an inner zone specific antigenic (IZAg) marker to give unequivocal identification of the adrenocortical cell types.

11 β-Hydroxylase mRNA was clearly shown to be expressed in the inner zones of the control rat adrenal cortex, and none was detected in the zona glomerulosa and medulla. The message was more abundant in the outer zona fasciculata. A similar pattern of distribution of 11β-hydroxylase mRNA was observed in adrenals from rats subjected to dietary sodium restriction, although the width of the negatively staining layer of zona glomerulosa was significantly increased. In rats treated with 100 μg ACTH for 1 day, the number of cells expressing 11β-hydroxylase mRNA was increased, especially in the zona reticularis. With continued ACTH treatment, 11β-hydroxylase mRNA was found in the region of the zona glomerulosa, and after 3 and 5 days of ACTH treatment cells expressing 11β-hydroxylase mRNA extended to the connective tissue capsule. At this time there was a significant reduction in the total expression of the message compared with the controls.

These results suggest that the presence of 11β-hydroxylase in the zona glomerulosa cells is not essential for the late pathway for aldosterone biosynthesis from deoxycorticosterone. Like IZAg, the distribution of 11β-hydroxylase mRNA after prolonged ACTH treatment provides further evidence to support the hypothesis that ACTH increases the conversion of zona glomerulosa to zona fasciculata-like cells.

Journal of Endocrinology (1993) 139, 301–306
Fragments of chromogranin A are present in the urine of patients with carcinoid tumours: development of a specific radioimmunoassay for chromogranin A and its fragments
Authors: Stridsberg M. , Hellman U. , Wilander E. , Lundqvist G. , Hellsing K. , Öberg K.
Published: December 2008

Chromogranin A is a well-known protein constituent in granules of neuroendocrine cells. It is also known that plasma levels of chromogranin A increase considerably in patients with neuroendocrine tumours and thus chromogranin A is used as a marker for these tumours. In the present study, we have shown that fragments of chromogranin A are excreted into the urine in some patients with carcinoid tumours. The chromogranin A molecule appeared in the urine N-terminally cleaved at amino acid positions 116 and 210, which are previously reported cleavage sites of the molecule. The fragments identified were mainly of about 35 kDa in size. The unprocessed chromogranin A molecule was not excreted in the urine. Five out of 40 patients excreting the fragments had slight tubular dysfunction in the kidneys. We also showed that these renally excreted split products of chromogranin A were immunogenic and could be used for production of antibodies against chromogranin A. These antibodies were used both for immunocytochemistry and for the development of a specific and sensitive radioimmunoassay for chromogranin A and its fragments. Measurements of plasma chromogranin A by radioimmunoassay appeared to be a better marker for tumour growth than were measurements of chromogranin A in the urine.

Journal of Endocrinology (1993) 139, 329–337
Investigation of the ontogenetic patterns of rat hypothalamic dopaminergic neurone morphology and function in vitro
Authors: Murray H. E. , Gillies G. E.
Published: December 2008

Using fetal rat hypothalamic cells in primary culture maintained in a serum-free defined medium we have investigated the morphological and functional development of the dopamine (DA)-containing neurones intrinsic to the hypothalamus.

Immunocytochemical studies demonstrated the presence of three morphologically distinct subtypes of tyrosine hydroxylase-immunopositive neurones. On day 3 in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two subtypes persisted to later days in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo.

Biochemical studies employing [3H]DA demonstrated a time- and temperature-dependent uptake mechanism within the cultures which was significantly attenuated by the uptake inhibitors benztropine and nomifensine in a dose-dependent manner. [3H]DA was also released under both basal and 56 mmol K+/l-stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The amount of [3H]DA accumulated and released by the cultured cells increased with the age of the culture, suggesting functional maturation of the DA-containing neurones within this preparation.

The role of oestradiol-17β in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of [3H]DA and by direct measurement of endogenously synthesized DA using high-performance liquid chromatography coupled with electrochemical detection. Both uptake and release of [3H] and release of endogenous DA were significantly modulated by the concentration of steroid in the defined medium.

These results demonstrate that hypothalamic dopaminergic neurones, when maintained in primary culture, undergo morphological and functional maturation which have several correlates in vivo. In addition, we have demonstrated that at least one sub-population of dopaminergic neurones within this preparation is responsive to oestradiol-17β.

As DA is considered to be a vital component in the regulation of neuroendocrine activity we suggest that this model is valuable for the investigation of the functional development of the DA systems of the hypothalamus and the relationship existing between neurotransmitters, neuropeptides and neuroactive steroids.

Journal of Endocrinology (1993) 139, 403–414
A modulatory role of endogenous opioids on prolactin secretion at the end of pregnancy in the rat
Authors: Soaje M , Deis R P
Published: December 2008

It is well known that the fall in serum progesterone concentrations during late pregnancy induces prolactin secretion in rats. On day 19 of pregnancy, administration of 10 mg of the antiprogesterone RU-486/kg induced a small but significant increase in serum prolactin. A lower dose (2 mg/kg) was not effective. Administration of naloxone (2 mg/kg) to pregnant rats on day 19 of pregnancy did not modify circulating prolactin but, after RU-486 treatment, a notable increase in serum prolactin was obtained 30 min after naloxone was given. The lack of effect of naloxone-methobromide in pregnant rats pretreated with RU-486 may indicate that the opioid-induced prolactin suppression acts centrally, most probably at the hypothalamic level. During day 21 of pregnancy, the time-course of prolactin secretion, measured at 0900, 1400, 1900 and 2200 h, was inversely correlated with circulating progesterone levels. At 0900 h, serum prolactin was very low with high serum progesterone concentrations but a significant increase in serum prolactin occurred at 2200 h; this was coincident with a significant decrease in the steroid. The stimulatory effect of naloxone on prolactin secretion was clearly dependent on the circulating progesterone level. Thus, at 1900 h of day 21, naloxone induced a significant increase in serum prolactin but, at 2200 h, the opioid antagonist dramatically enhanced the circulating level of prolactin. The serum prolactin increase induced by naloxone at 1900 h was prevented by the s.c. administration of 5 mg progesterone given 7 h earlier. Similarly, the large increase in serum prolactin levels at 1800 h on day 19 of pregnancy, after administration of RU-486 plus naloxone, was completely abolished by treatment with CB154. The lack of effect of RU-486 and naloxone on serum prolactin levels in virgin rats on the day of pro-oestrus demonstrates that the effect of naloxone on prolactin in pregnant rat is peculiar to the end of pregnancy.

In conclusion, the attenuation of the central inhibitory action of progesterone facilitates the release of prolactin which is dramatically enhanced by naloxone treatment. These results provide an important new insight into the existence of a neuromodulatory regulation of prolactin secretion by the opioid system showing a paradoxical opioid-induced prolactin suppression at the end of pregnancy.

Journal of Endocrinology (1994) 140, 97–102
Lipopolysaccharide modulation of eicosanoid and corticotrophin-releasing hormone release from rat hypothalamic explants and astrocyte cultures in vitro: evidence for the involvement of prostaglandin E2 but not prostaglandin F2α and lack of effect of nerve growth factor
Authors: Pozzoli G , Costa A , Grimaldi M , Schettini G , Preziosi P , Grossman A , Navarra P
Published: December 2008

Bacterial lipopolysaccharide (LPS) and prostaglandins (PG) E2 and F2α are putative activators of the hypothalamo-pituitary-adrenal axis. Certain of the biological effects of LPS may be mediated by cytokines such as interleukin-1β (IL-1β), while IL-1β itself may operate via induction of the prostaglandins and/or nerve growth factor (NGF). As IL-1β stimulates the release of corticotrophin-releasing hormone (CRH) from acute rat hypothalamic explants directly, the effects of these substances on the release of CRH in vitro were investigated in short- and medium-term (20 and 60 min) incubations. The effect of LPS on the release of PGE2 and PGF2α from these explants, as well as from cortical astrocyte cultures, was also studied.

LPS did not modify the release of CRH, PGE2 or PGF2α in 20-min incubations. In 60-min incubations, LPS stimulated the release of PGE2, whereas the release of CRH was weakly, but significantly, reduced; PGF2α was not altered. PGE2 significantly stimulated CRH release in the 60-min but not in the 20-min experiments. This effect appeared to be selective for PGE2, since PGF2α did not modify CRH release, alone or in combination. LPS also selectively released PGE2 but not PGF2α from cortical astrocyte cultures after 24-h incubation. NGF had no effect on the release of explant CRH, regardless of the length of incubation.

It was concluded that neither LPS nor NGF acutely stimulate the hypothalamo-pituitary-adrenal axis by a direct action on hypothalamic CRH, while hypothalamic PGE2 may mediate, at least in part, certain of the neuroendocrine responses to LPS.

Journal of Endocrinology (1994) 140, 103–109
Long-term changes of somatotrophic function induced by deprivation of growth hormone-releasing hormone during the fetal life of the rat
Authors: Cella S G , Locatelli V , Broccia M L , Menegola E , Giavini E , De Gennaro Colonna V , Torsello A , Wehrenberg W B , Müller E E
Published: December 2008

We have studied the effects of intra-amniotic administration of an anti-GH-releasing hormone serum (GHRH-Ab) on day 16 of fetal life in the rat, when the ontogenetic development of the GHRH neuronal system occurs. Control animals received normal rabbit serum. Following delivery, body weight was monitored for the next 30 days as an index of somatic growth, and the following indices of somatotrophic function were determined: plasma and pituitary GH, pituitary GH mRNA, hypothalamic GHRH and somatostatin mRNA, and the in vivo GH responsiveness to GHRH. At birth, GHRH-Ab-treated rats had a body weight that was equivalent to that of control rats but, starting from postnatal day 6 up to day 30, they had a significantly reduced body weight. Pituitary weight, the absolute pituitary GH content and GH mRNA levels were lower in experimental compared with control rats, while pituitary GH concentrations were similar in the two groups, thus implying that there was a defect, not only in GH synthesis, but also in GH release. In agreement with this theory, basal GH levels and GHRH-stimulated GH secretion were reduced in GHRH-Ab-treated rats but, in contrast, hypothalamic regulation of GH secretion appeared to be working in these rats as they were still able to respond to the low plasma GH by increasing GHRH and decreasing somatostatin mRNA levels. These findings indicate that deprivation of GHRH during fetal life induces long-lasting changes of growth rate and somatotrophic function. In addition, when comparing these changes with those in rats given GHRH-Ab postnatally, it would appear that deprivation of GHRH affects different regulatory levels of the hypothalamo-pituitary-somatotroph axis depending on when the deprivation occurs.

Journal of Endocrinology (1994) 140, 111–117