bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of adrenal steroidogenesis by adrenaline expression of cytochrome P450 genes
Authors: Güse-Behling H. , Ehrhart-Bornstein M. , Bornstein S. R. , Waterman M. R. , Scherbaum W. A. , Adler G.
Published: December 2008

The effect of adrenaline on the secretion of cortisol and cyclic AMP (cAMP) and on the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450scc), 17α-hydroxylase cytochrome P450 (P45017α), 21-hydroxylase cytochrome P450 (P450c21) and 11β-hydroxylase cytochrome P450 (P45011β) was studied in bovine adrenocortical cells in primary culture and compared with the effects of ACTH. Treatment of cultured cells with adrenaline (1–100 μmol/l) showed a biphasic response in cortisol release over 1–24 h. Concentration of cAMP in the culture media increased from a basal level of <0·06 pmol/dish to a maximal level of 40·14 ± 8·9 pmol/dish with a half-maximal release of 20·07 pmol cAMP/dish in the medium reached 1·2 h after treatment with 10 μmol adrenaline/l. This stimulation resulted in an uniform increase in the levels of all four P450 mRNAs as revealed by Northern blot analysis. Increasing doses of adrenaline produced a maximal mRNA accumulation at a concentration of 10 μmol adrenaline/l. Incubation of the cells with 10 μmol adrenaline/l for 1–24 h produced a biphasic time-course with a half-maximal stimulation after about 5–6 h. Maximal stimulation with ACTH (100 nmol/l) caused different accumulations of the four mRNAs: P450sec mRNA increased twice as much and P45017α mRNA six times as much as the accumulation of P450c21 mRNA and P45011β mRNA, which was about ten-fold over basal values. Propranolol totally blocked the stimulatory effect of adrenaline but not the effect of ACTH. Our data suggest that the stimulatory effect of adrenaline upon de-novo cortisol formation is regulated in part at the transcriptional level in a dose- and time-dependent manner. The regulation requires β-adrenergic receptors and is due to a cAMP-mediated increase in the accumulation of all four P450 mRNAs.

Journal of Endocrinology (1992) 135, 229–237
Antiuterotrophic effects of a pure antioestrogen, ICI 182,780: magnetic resonance imaging of the uterus in ovariectomized monkeys
Authors: Dukes M. , Miller D. , Wakeling A. E. , Waterton J. C.
Published: December 2008

ICI 182,780 is a potent specific pure antioestrogen which may offer advantages in breast cancer treatment compared with partial agonists like tamoxifen. To characterize further the potency and efficacy of ICI 182,780, its effects on the uterus of ovariectomized, oestrogen-treated monkeys (Macaca nemestrina) have been measured using magnetic resonance imaging (MRI). Quantitative MRI allows accurate non-invasive repetitive measurements of endometrial and myometrial volume following hormonal treatments, using each animal as its own control. Single i.m. injections of a long-acting oil-based formulation of ICI 182,780 sustained blockade of oestradiol action on the monkey uterus in a dose-dependent manner for 3–6 weeks. Repeated injections of 4 mg ICI 182,780/kg at 4-weekly intervals provided increasingly effective blockade of uterine proliferation. In a short-acting formulation, ICI 182,780 also completely blocked the trophic action of oestradiol, administered concurrently, in ovariectomized monkeys. Similarly, ICI 182,780 caused involution of the uterus stimulated by prior treatment with oestradiol. The rate and extent of uterine involution in monkeys treated with ICI 182,780 was similar to that seen following oestrogen withdrawal. These studies demonstrate that ICI 182,780 is a fully effective pure antioestrogen in a primate.

Journal of Endocrinology (1992) 135, 239–247
A comparison of the uterotrophic response in cyclic and ovariectomized normotensive and spontaneously hypertensive rats
Authors: Kerr M. B. , Marshall K. , Senior J.
Published: December 2008

The uterotrophic response in the normotensive (CD) and the hypertensive (SHR) rat was compared in intact cyclic rats and in ovariectomized rats given oestradiol. The parameters measured were blood flow, and uterine wet and dry weights. In the cyclic animals blood flow to the oestogen target tissue varied throughout the oestrous cycle, peak flows being achieved at pro-oestrus; in the SHR rat, however, the pro-oestrous maximum was significantly attenuated compared with the CD rat. Uterine wet and dry weights were similar.

The temporal response to oestradiol in ovariectomized rats showed that in the CD rat the hyperaemic response peaked earlier than in the SHR rat, significant changes in terms of increased water imbibition also occurred more quickly in the CD strain. In both strains, uterine dry weight was the last parameter to be significantly increased, the maximum weight being attained more quickly in the SHR rat.

The results of this study indicated that it is the blood flow to the oestrogen target tissues of the uterus and vagina that is most susceptible to change with strain of rat.

Journal of Endocrinology (1992) 135, 263–269
Fine structure of adrenal cortex in rats harbouring a medullary thyroid carcinoma transfected with a corticotrophin-releasing hormone cDNA expression vector
Authors: Feinmesser M. , Asa S. L. , Kovacs K. , Low M. J.
Published: December 2008

We report the light microscopic, transmission and scanning electron microscopic features of the adrenal cortices in rats bearing a medullary thyroid carcinoma cell line transfected with a corticotrophin-releasing hormone (CRH) cDNA expression vector. The animals had elevated CRH, ACTH and corticosterone blood levels, involuted thymuses and markedly enlarged adrenal glands with prominent lipid-depleted cortices and dilated congested capillaries, similar to those of animals treated with ACTH. Using electron microscopy it was found that the enlarged fasciculata and reticularis zones were composed of large, compact cells with abundant smooth endoplasmic reticulum, prominent Golgi complexes, increased number of large mitochondria with focal loss of cristae and cavitation of the internal compartments, numerous lysosomes and prominent elongated microvilli. In addition, small cytoplasmic fragments were seen within the capillary lumina; these structures resembled microvilli that were apparently detached from adrenocortical cells and entered the blood stream via discontinuous endothelium of dilated capillaries. By scanning electron microscopy it was found that the cells had bulging surfaces with scattered pits and numerous long microvilli pointing in different directions.

This animal model allows analysis of the effects of protracted CRH excess resembling tumoural CRH-dependent Cushing's syndrome in human patients. Our findings call attention to the role of microvilli in adrenocortical secretion. The increased number and size of microvilli has been thought to lead to an increase in the surface area of adrenocortical cells, thereby facilitating hormone discharge. The detachment of microvilli from adrenocortical cells may represent a form of apocrine secretion and may contribute to hypercorticosteronaemia in CRH excess.

Journal of Endocrinology (1992) 135, 271–277
A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry
Authors: Okamura H. , Yamamoto K. , Hayashi S. , Kuroiwa A. , Muramatsu M.
Published: December 2008

A rat oestrogen receptor-β-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct. Scatchard analysis of [3H]oestradiol-17β binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1·49 nmol/l. The relative molecular weight (Mr) of the fusion protein was determined as 180 000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor.

The protein was isolated by means of SDS-PAGE and subsequent electroblotting. By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit. Binding of [3H]oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner. The antibody was also able to recognize the oestrogen receptor occupied by [3H]oestradiol. Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone. In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of Mr 67 000, the size of the oestrogen receptor, was detected by the antibody. Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain. The immunoreactivity was restricted exclusively to cell nuclei in both tissues.

These results demonstrate that the polyclonal antibody obtained in the present study was specific to the oestrogen receptor, and that it would be a powerful tool to detect and analyse the receptors in various target tissues for oestrogen.

Journal of Endocrinology (1992) 135, 333–341
Inhibition of bovine adrenocortical steroidogenesis by benzodiazepines: a direct effect on microsomal hydroxylation or an inhibition of calcium uptake?
Authors: Thomson I. , Fraser R. , Kenyon C. J.
Published: December 2008

We have previously reported that benzodiazepines inhibit microsomal steroid hydroxylases. We have now studied their effects at much lower drug concentrations and have also addressed the suggestion that benzodiazepines alter cellular calcium metabolism.

We investigated the in-vitro effects of midazolam on microsomal steroid hydroxylation by measuring basal and ACTH-stimulated cortisol and 17α-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3·4 μmol midazolam/1 while ACTH-stimulated production required 13·6 μmol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13·6 μmol midazolam/l for basal and 6·8 μmol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6·8 μmol/l and additonal effects on 17α-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 μmol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 μmol/l).

Both midazolam and diazepam inhibited K+-stimulated aldosterone synthesis, with IC50 values of 1·2 μmol/l and 0·8 μmol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11β-hydroxyprogesterone as substrate, the KI for the inhibition of aldosterone synthesis by midazolam was 54 μmol/l. Potassium stimulates aldosterone biosynthesis at least partly by changing intracellular free calcium levels. To investigate possible antagonistic effects of benzodiazepines on calcium metabolism, we measured 45Ca uptake in the presence of midazolam. Both basal (P < 0·01) and K+-stimulated 45Ca uptake (P < 0·05) were inhibited by the drug although the effects of K+ were not completely abolished. Comparison of the dose-dependent effects of midazolam on basal 45Ca uptake in cell suspensions prepared from different areas of the adrenal cortex indicated that zona glomerulosa cells are more sensitive to midazolam.

We confirm that benzodiazepines at low concentrations have a direct effect on microsomal steroid hydroxylase enzymes in vitro and postulate that the greater sensitivity to benzodiazepines of K+-stimulated aldosterone synthesis, when compared with either ACTH-stimulated cortisol synthesis or conversion of 21-deoxycortisol to cortisol, may be explained by additional effects of these drugs on plasma membrane calcium transport.

Journal of Endocrinology (1992) 135, 361–369
Restoration of juvenile baseline growth hormone secretion with preservation of the ultradian growth hormone rhythm by continuous delivery of growth hormone-releasing factor
Authors: Vasilatos-Younken R. , Tsao P. H. , Foster D. N. , Smiley D. L. , Bryant H. , Heiman M. L.
Published: December 2008

The ability of continuously delivered GH-releasing factor (GRF) to enhance GH secretion while maintaining the normal ultradian GH rhythm was investigated. Synthetic human GH-releasing factor (hGRF(1–44)NH2) was continuously infused for 4 days by means of i.v. catheters to 11-week-old broiler chickens. At this age, overall endogenous GH secretion is low, and baseline GH is barely detectable. Six birds per treatment received vehicle (control), 0·324 mg hGRF(1–44)NH2/kg body weight per day (low dose) or 3·24 mg hGRF(1–44)NH2/kg body weight per day (high dose). After 4 days of GRF conditioning, concurrent with continued GRF infusion, serial blood samples were removed via atrial catheters at 15-min intervals for 6 h and GH plasma profiles determined.

High dose GRF significantly increased GH plasma concentrations over tenfold compared with controls; however, most of this increase reflected an increase in basal GH, which was reinstated to juvenile baseline levels. Augmentation of pulse amplitude above this increased baseline was not proportionately as high, and failed to reach juvenile levels. The ultradian rhythm of GH was not altered by continuous GRF administration.

Both low and high dose GRF treatments resulted in significant enlargement of the anterior pituitary gland. Total pituitary GH mRNA levels, although elevated over twofold by GRF treatment, were not significantly different from controls. Measures of plasma GH magnitude (overall and baseline mean, and peak amplitude) were significantly correlated with pituitary GH mRNA for control birds, but were not correlated for GRF treatments.

Feed intake was markedly depressed (33%) on the high dose GRF treatment, in conjunction with total inhibition of body weight gain over the 4-day period of administration. Longitudinal bone growth and width of the epiphyseal growth plate were also significantly reduced by high dose GRF treatment, probably reflecting the reduced level of nutrient intake, despite high circulating concentrations of GH.

Journal of Endocrinology (1992) 135, 371–382
Intracellular pH regulation in human thyrocytes: evidence of both Na+/H+ exchange and Na+-dependent Cl−/HCO−3 exchange
Authors: Kayser L. , Høyer P. E. , Perrild H. , Wood A. M. , Robertson W. R.
Published: December 2008

The present study was undertaken to investigate the regulation of intracellular pH in human thyroid epithelial cells and to characterize the kinetics of the acid-extruding processes operating in the absence and presence of HCO−3, Na+ and Cl−. A dynamic technique of dual excitation microfluorimetry and the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein was employed.

The intracellular pH was 7·01 ± 0·27 (n = 29) and 6·94 ± 0·25 (n = 54) in the absence and presence of HCO−3 respectively. Both in the absence and presence of HCO−3, the recovery from intracellular acid loads was not only due to an Na+/H+ exchange, but also to an Na+-dependent HCO−3/Cl− exchange. In alkaline conditions caused by NH4Cl pulsing, an HCO−3/Cl− exchange was also found. The cells in HCO−3 responded with a wide range of maximal hydrogen efflux rates in experiments where cells were either pretreated with 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid or incubated with amiloride. The heterogeneity might be due to subpopulations of thyrocytes in different metabolic states or at different points in the cell cycle.

It is concluded that recovery from intracellular acidification in human thyroid cells is due to both Na+/H+ exchange and Na+-dependent Cl−/HCO−3 exchange even in nominally HCO−3-free conditions, and that recovery from intracellular alkalinization is due to a Cl−/HCO−3 exchange which needs to be characterized further.

Journal of Endocrinology (1992) 135, 391–401
Prepubertal reduction of the ovarian follicle population by combined LH-releasing hormone antagonist treatment and unilateral ovariectomy influences follicle characteristics but not the ovulation rate at first oestrus
Authors: van Cappellen W. A. , van Leeuwen E. C. M. , Kramer P. , Meijs-Roelofs H. M. A.
Published: December 2008

The effect on first ovulation of the massive reduction of the total pool of ovarian follicles during the infantile and late juvenile period was studied in rats. Treatment with an LH-releasing hormone antagonist (LHRH-A) during infancy (5 mg/kg body weight on days 6, 9, 12 and 15 of life) was combined with unilateral ovariectomy performed on either day 15 (early ULO) or 2–5 days before the expected day of first ovulation (late ULO). Rats were killed on the day of first or second oestrus, when blood was collected and the (remaining) ovaries were prepared for differential counting of follicles and corpora lutea. In addition, blood was sampled 8 h after ULO and the ovaries studied histologically in the group of rats which were unilaterally ovariectomized 2–5 days before first ovulation.

The time of first ovulation was not influenced by treatment with LHRH-A, early or late ULO, or a combination of LHRH-A treatment and ULO. Ovulation rate after LHRH-A treatment was decreased, but was still within the normal range in intact rats and in early ULO rats compared with saline-treated controls.

Serum FSH concentrations 8 h after ULO performed 2–5 days before first ovulation were similar in saline- and LHRH-A-treated rats (845 ± 59 and 801 ± 99 (s.e.m.) μg/l respectively) and had increased compared with intact controls (216 ± 15 μg/l).

Treatment with LHRH-A resulted in a reduction of more than 50% in healthy and atretic follicles, and late ULO reduced the number of healthy follicles even further. In saline-treated rats late ULO decreased the rate of atresia, but in LHRH-A-treated rats atresia was not reduced further by (late or early) ULO.

It is concluded that even after massive reduction of the pool of ovarian follicles by early LHRH-A treatment combined with late or early ULO, the timing of the first ovulation was normal and ovulation rates, although somewhat lower in some LHRH-A-treated rats, were within the normal range.

Journal of Endocrinology (1992) 135, 439–446
Catecholamines released from local adrenergic axon terminals are possibly involved in fine tuning of steroid secretion from zona glomerulosa cells: functional and morphological evidence
Authors: Vizi E. S. , Tóth I. E. , Szalay K. Sz. , Windisch K. , Orsó E. , Szabó D. , Vinson G. P.
Published: December 2008

The effect of supramaximal electric field stimulation on [3H]noradrenaline (NA) release and hormone production by rat adrenal capsule-glomerulosa preparations was studied using a microvolume perfusion system. A substantial proportion (about 20%) of nerve endings (varicosities) were observed close to zona glomerulosa cells, and about half of them appeared to be catecholaminergic, as judged by the chromaffin reaction of the synaptic vesicles studied at electron microscopic level. In tissue, preloaded with [3H]NA, the release of NA in response to electrical stimulation was frequency-dependent. Reserpinization, calcium removal or inhibition of Na+ influx by tetrodotoxin completely blocked NA release by field stimulation, indicating that the release resulted from axonal activity and is of vesicular origin. Neither the α2-adrenoceptor agonist xylazine nor the muscarine-receptor agonist oxotremorine affected the stimulation-evoked release of [3H]NA, suggesting that, in contrast with other neurones present in the central nervous system or in the peripheral autonomic nervous system but like those in the median eminence, these axon terminals contained few presynaptic modulatory receptors. The NA (10·20 ± 1·79 (s.e.m.) μg/g, n = 9), adrenaline (24·38 ± 5·50 μg/g, n = 9) and dopamine (0·35 ± 0·09 μg/g, n = 6) contents of the preparations were high, as determined by high-performance liquid chromatography. Our observations that the release and content of NA is high, and that a substantial proportion of catecholaminergic axon terminals lie in close proximity to zona glomerulosa cells (median value of the distance 300 nm) or to smooth muscle cells of the vessels, suggest that NA released from local adrenergic neurones without being presynaptically modulated may play an important role in finetuning both steroid production and/or blood flow through the gland, itself a powerful modulator of the adrenocortical response. This local modulating effect of NA may be especially significant when sympathetic activity is enhanced.

Journal of Endocrinology (1992) 135, 551–561
Evidence for lipopolysaccharide binding in human granulosa-luteal cells
Authors: Sancho-Tello M. , Chen T.-Y. , Clinton T. K. , Lyles R. , Moreno R. F. , Tilzer L. , Imakawa K. , Terranova P. F.
Published: December 2008

We investigated whether human granulosa-luteal (GL) cells exhibited lipopolysaccharide (LPS)-binding protein, and the response of follicular aspirate cells to LPS in vitro. Follicular aspirates taken from a human in-vitro fertilization and gamete intrafallopian-tube transfer programme were subjected to Percoll gradients in order to isolate an enriched population of GL cells. GL cells exhibited specific LPS-binding protein, detected by autoradiography of the cellular lysate on SDS-PAGE after the cells were specifically labelled with a radioiodinated, photoactivable and reducible LPS derivative. LPS binding to the cells was also detected by the appearance of immunofluorescence associated with the cellular membrane when incubated with a fluorescent conjugated LPS receptor antibody. Ninety-four per cent of the cells exhibiting immunofluorescent LPS-binding protein were also positive for the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase, as detected by cytochemistry. In order to detect a response to LPS, the enriched population of GL cells were cultured in vitro in the presence or absence of LPS; after 16 h of culture, tumour necrosis factor-α (TNF) mRNA was detected by reverse transcription-polymerase chain reaction and Southern blot analysis of the amplified cDNA. The expression of TNF mRNA was enhanced when the cells were cultured in the presence of LPS, which also significantly enhanced TNF secretion into the media during the 16-h period. These results reveal that GL cells exhibit LPS-binding protein and thus increased TNF secretion occurs in response to LPS in follicular aspirate cells. The source of ovarian TNF may be leukocytes, macrophages and/or GL cells.

Journal of Endocrinology (1992) 135, 571–578
Inhibition of corticosteroid production by sodium pentobarbitone in rat adrenocortical preparations
Authors: Whitehouse B. J. , Purdy S. J. , Abayasekara D. R. E.
Published: December 2008

It is possible that some of the effects of sodium pentobarbitone on the hypothalamo-pituitary-adrenal axis in the intact animal may be attributable to direct actions on the adrenal cortex. The effects of the barbiturate on steroid production by rat adrenal preparations in vitro have therefore been examined. In zona glomerulosa cells, pentobarbitone inhibited basal steroid production in a dose-related fashion. For aldosterone and corticosterone, the doses required for 50% inhibition of production (IC50) were 1·2 mmol pentobarbitone/l and 3·7 mmol/l respectively. Steroidogenesis was inhibited at lower levels of pentobarbitone in the presence of 1 nmol ACTH/l (IC50 = 0·5 mmol pentobarbitone/l for aldosterone and 2·2 mmol/l for corticosterone). In zona fasciculata/reticularis cells, production of corticosterone was similarly reduced with an IC50 of 2·8 mmol pentobarbitone/l for basal production and 1·3 mmol/l for ACTH-stimulated production. The dose-related increases in corticosterone production produced by ACTH (0·1–1000 pmol/l) or dibutyryl cyclic AMP (0·1–1·0 mmol/l) were also eliminated in the presence of 2 mmol pentobarbitone/l. The effects of pentobarbitone (1–4 mmol/l) on the production of pregnenolone and deoxycorticosterone (DOC) were also studied. In zona fasciculata/reticularis cells, the responses of both pregnenolone and DOC were bell-shaped with increases at 1 mmol pentobarbitone/l, which fell back to control levels at 4 mmol pentobarbitone/l. Stimulation of DOC, accompanied by decreases in aldosterone and corticosterone production, was also seen in zona glomerulosa cells at 1 mmol pentobarbitone/l.

The effect of 1 mmol pentobarbitone/l on the conversion of 22(R)-hydroxycholesterol (5-cholestene-3β,22(R)-diol), pregnenolone, progesterone and DOC to corticosterone and aldosterone by zona glomerulosa preparations was studied. There was a comparable reduction in the conversion of these precursors (2 μmol/l) to aldosterone with yields decreased to 20–30% of those found in the absence of pentobarbitone. The dose required for 50% reduction of the conversion of progesterone (2 μmol/l) to aldosterone was 0·55 mmol pentobarbitone/l and for corticosterone the dose was 1·75 mmol pentobarbitone/l.

The results obtained show that pentobarbitone is an effective inhibitor of corticosteroid biosynthesis in rat adrenal cells, and suggest that its effects are brought about by inhibition of cytochrome P450-mediated hydroxylations.

Journal of Endocrinology (1993) 136, 75–83
β-Endorphin in sows during late pregnancy: effects of cloprostenol and oxytocin on plasma concentrations of β-endorphin in the jugular and uterine veins
Authors: Aurich J. E. , Dobrinski I. , Parvizi N.
Published: December 2008

β-Endorphin was measured in the plasma of pigs during late pregnancy and at different stages of the oestrous cycle. In pregnant animals, β-endorphin secretion from uteroplacental tissues into the maternal circulation and the possible effects of oxytocin and the prostaglandin F2α (PGF2α) analogue cloprostenol on β-endorphin release were determined. Plasma β-endorphin concentrations in pregnant sows were significantly higher than in non-pregnant pigs. However, there were no significant changes in β-endorphin values throughout the oestrous cycle. Because the increase in plasma β-endorphin concentrations had occurred before luteolysis and onset of labour it could not be attributed to the stress of parturition. The surgical intervention of a laparotomy increased β-endorphin release into peripheral plasma. Cloprostenol but not oxytocin caused an immediate increase in plasma β-endorphin concentrations. At parturition, endogenous PGF2α may be involved in the regulation of β-endorphin secretion. Concentrations of β-endorphin in the jugular and uterine vein plasma were not significantly different, and so it would appear that β-endorphin in the plasma of pregnant sows is not of uteroplacental origin. In conclusion, changes in the concentration of β-endorphin in peripheral plasma, associated with pregnancy but not the oestrous cycle, exist in pigs. Hence a physiological function of peripheral opioid peptides in the periparturient sow is feasible.

Journal of Endocrinology (1993) 136, 199–206
Regulation of plasma and tissue levels of insulin-like growth factor-I by nutrition and treatment with growth hormone in sheep
Authors: Hua K. M. , Ord R. , Kirk S. , Li Q. J. , Hodgkinson S. C. , Spencer G. S. G. , Molan P. C. , Bass J. J.
Published: December 2008

Tissue and plasma levels of insulin-like growth factor-I (IGF-I), and relative levels of liver IGF-I RNA, were measured in 6-month-old ewe lambs which were well fed (n = 10) or starved (n = 10) for 5 days. Half of each nutrition group was given daily (09.00 h) injections of human GH (hGH; 0·15 mg/kg body weight per day). Blood was sampled daily from 09.00 to 12.00 h at 15-min intervals through jugular vein catheters and the lambs were slaughtered 24 h after the fifth injection of hGH.

Tissue and plasma IGF-I was extracted using an acid-ethanol-cryo-precipitation technique and estimated by radioimmunoassay. Tissue IGF-I was corrected for retained plasma IGF-I using tissue and blood haemaglobin levels. Liver IGF-I RNA levels were monitored by in-situ hybridization.

Plasma IGF-I (nmol/l) was higher in both the fed group and the fed group given GH treatment. Tissue IGF-I from kidneys (nmol/kg) was also higher (P < 0·001) in the fed group. There was no significant difference in IGF-I concentrations in the muscle biceps femoris or liver between fed and starved lambs. Although GH treatment did not increase IGF-I levels in tissues significantly, IGF-I RNA levels in liver were increased (P = 0·02) in both fed and starved animals. The relative liver IGF-I RNA levels positively correlated with their corresponding tissue IGF-I levels in the fed group and the fed group given GH treatment. The lack of a significant IGF-I response to GH in tissues may be due to either the time at which tissues were sampled after the GH treatment or the dose of GH administered. However, the higher IGF-I concentrations in plasma and kidney from fed compared with starved animals and the positive correlations between liver IGF-I and IGF-I RNA levels suggest that tissue and plasma IGF-I is regulated by nutrition and GH, with nutrition playing a critical role in the regulation of tissue and plasma IGF-I in normal lambs.

Journal of Endocrinology (1993) 136, 217–224
FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta)
Authors: Arslan M. , Weinbauer G. F. , Schlatt S. , Shahab M. , Nieschlag E.
Published: December 2008

The present study was designed to investigate the relative contributions of FSH and testosterone in the initiation of testicular growth and function in primates. Four groups (n = 4/group) of juvenile rhesus monkeys (Macaca mulatta), 12–18 months old, were treated with vehicle, a highly purified human FSH preparation (hFSH; Fertinorm, 3 IU/kg per day), testosterone (testosterone enanthate, 125 mg/week) or FSH plus testosterone, for a period of 12 weeks. Compared with vehicle treatment, the administration of hormones significantly (P <0·05) increased testicular weight and volume, and the diameter of seminiferous tubules. The number of Sertoli cells per tubule cross-section also increased significantly (P <0·05). Numbers of Ad (dark) spermatogonia (reserve stem cells) were not significantly influenced by any treatment. In contrast, the numbers of Ap (pale) spermatogonia (renewing stem cells) were significantly (P <0·05) stimulated with hFSH and testosterone alone. Following the combined treatment, numbers of Ap spermatogonia were also higher compared with control but this effect did not attain statistical significance. In half of the animals in both testosterone-treated groups, a few prophase I spermatocytes were present. Inhibin concentrations reached adult levels in hFSH-treated groups but remained unaffected by testosterone. Conversely, testosterone failed to influence inhibin levels and, unlike hFSH, increased testicular androgen concentration and epididymal weights. Our observations suggest that hFSH and testosterone alone are capable of initiating testicular growth and gametogenesis in an immature primate. Both hormones probably act via activation of the proliferation of Ap spermatogonia, which are considered to be renewing stem cells within the testis. This study also suggests that androgens might be involved in the morphological differentiation of the immature primate Sertoli cell, whereas the secretory activity of the seminiferous tubule, in terms of peripheral inhibin levels, might be under the influence of FSH.

Journal of Endocrinology (1993) 136, 235–243
Insulin-like growth factor-binding protein-1, glucose tolerance and fetal growth in human pregnancy
Authors: Baldwin S. , Chung T. , Rogers M. , Chard T. , Wang H. S.
Published: December 2008

Two hundred Chinese primigravidae had 50 g 3-h oral glucose tolerance tests (OGTTs) twice in pregnancy; between 20 and 24 weeks and between 30 and 34 weeks of gestation. In 149 women, a single sample was taken for insulin-like growth factor-binding protein-1 (IGFBP-1) measurement 0, 1, 2 or 3 h after the glucose load at both visits; in 55 women IGFBP-1 levels were estimated in all four OGTT samples. Fetal growth was assessed by ultrasound performed at the first and second visit and, if possible, at term, and by anthropometry of the neonate. Cord serum IGFBP-1 was measured in 144 of the babies. Mothers who developed gestational diabetes were excluded.

Maternal levels of IGFBP-1 were inversely related to glucose levels at 0, 1 and 2 h in the third trimester of pregnancy. IGFBP-1 measured at 1 h in an OGTT increased between the second and third trimester. There was an inverse correlation between maternal IGFBP-1 measured in the second trimester and all fetal measurements at that time, and with most neonatal measurements and birthweight. Levels of IGFBP-1 in the third trimester were inversely correlated to neonatal abdominal circumference, skinfold thickness and birthweight. Cord blood IGFBP-1 was inversely related to growth of abdominal circumference. The strongest inverse relationship was between IGFBP-1 and maternal weight. Fasting glucose in the second trimester was positively correlated to fetal subcutaneous fat and growth of abdominal circumference. In the third trimester it was related to fetal abdominal circumference, the growth of abdominal circumference, birthweight and neonatal skinfold thickness. In a multiple regression analysis, both glucose and IGFBP-1 were shown to be determinants of birthweight. It is concluded that IGFBP-1 levels are related to glucose tolerance in pregnancy, and that both IGFBP-1 and glucose have a role in determining birthweight.

Journal of Endocrinology (1993) 136, 319–325
The effect of free fatty acids on the in-vitro binding of testosterone in human plasma
Authors: Diver M. J.
Published: December 2008

The effect of supraphysiological levels of free fatty acids (FFA) on the binding of testosterone to sex-hormone binding globulin (SHBG) and on non-SHBG binding in both male plasma and plasma from pregnant women was studied. Six FFAs were added to plasma as individual acids. No alteration in testosterone binding to SHBG could be demonstrated with any of the FFAs in either male plasma or plasma from pregnant women. When the same plasma was heated to destroy SHBG binding, a highly significant (P <0·01) increase in non-SHBG binding was seen in both male plasma and plasma from pregnant women when the unsaturated FFAs oleic, linoleic and linolenic acids were added. No significant difference was demonstrated with the saturated FFAs, palmitic, stearic and arachidic acids.

Journal of Endocrinology (1993) 136, 327–330
Serum levels of adrenal androgens up to adrenarche in Peruvian children living at sea level and at high altitude
Authors: Goñez C. , Villena A. , Gonzales G. F.
Published: December 2008

Serum levels of adrenal androgens were measured in children of both sexes living at sea level and at high altitude using a cross-sectional design. The levels were compared in relation to age at each altitude (150 m and 3400 m above sea level), and the first significant increase in mean levels compared with values at preceding ages was assessed and defined as adrenarche.

A total of 118 boys and 95 girls aged 6–12 years living at low altitude and 95 boys and 104 girls aged 7–15 years living at high altitude, all of them attending public schools, were studied. Serum dehydroepiandrosterone, dehydroepiandrosterone sulphate and androstenedione were measured by radioimmunoassay and height and weight by standard anthropometric techniques. There were two significant increases in serum levels of adrenal androgens, the first observed in children between 6 and 8 years at sea level, and between 7 and 9 years at high altitude, and the second in children between 10 and 12 years at sea level and between 12 and 15 years at high altitude. Serum adrenal androgen levels increased earlier in children at sea level than at high altitude.

It is concluded that adrenarche occurs later at high altitude than at sea level.

Journal of Endocrinology (1993) 136, 517–523
ENDOTHELIN STIMULATES TESTOSTERONE SECRETION BY RAT LEYDIG CELLS
Authors: Conte D. , Questino P. , Fillo S. , Nordio M. , Isidori A. , Romanelli F.
Published: December 2008

The present study was designed to evaluate the effects of endothelin (ET) on rat testicular steroidogenesis in vitro and the involvement of prostaglandins (PG) and extracellular calcium in its mechanism of action. To this purpose we examined the effects of ET-1 and ET-3 on basal testosterone secretion, the influence of ET-1 on PGE2, release, the interaction of ET-1 and ET-3 with human chorionic gonadotrophin (hCG) and the interference of indomethacin (an inhibitor of cycloxygenase) and nifedipine (a calcium-channel blocker) in purified rat Leydig cells. The data indicate that ET-1 and ET-3 stimulate basal and hCG-induced testosterone production although the effects of ET-3 were less marked. In addition, a concomitant release of PGE2, was observed after exposure to ET-1. A sinergistic interaction between ET-1 and hCG in stimulating testicular steroidogenesis was revealed. Indomethacin was ineffective in modifying ET-1 evoked testosterone output, while in the presence of nifedipine the stimulatory effect of ET-1 was completely abolished. Since it has been shown by others that ET-1 is produced by rat Sertoli cells and specific binding sites are present in Leydig cells, the results of our study indicate that such a peptide may be regarded as a new paracrine factor able to influence steroidogenesis in Leydig cells. The action of ET-1 requires the activity of voltage-operated Ca2+ channels, while PGE2 activation is not essential for its steroidogenic effect.
Norethindrone acetate only partially protects the skeleton of rats treated with the LHRH agonist buserelin from oestrogen-deficiency osteopaenia
Authors: Goulding A. , Gold E.
Published: December 2008

In women with endometriosis there is concern that therapeutic use of LH-releasing hormone (LHRH) analogues, to lower ovarian oestrogen production and control endometrial hyperplasia, leads to unwanted oestrogen-deficiency bone loss. We have developed an animal model of this LHRH-mediated oestrogen-deficiency bone loss in the rat, using buserelin. The aim was to use this model to determine whether the progestogen, norethindrone acetate, could counter oestrogen-deficiency bone loss associated with prolonged treatment with the LHRH agonist buserelin. Four groups of animals which had their bones labelled with 45Ca were studied for 4 weeks: group A, control; group B, buserelin treated; group C, norethindrone acetate treated; group D, norethindrone acetate + buserelin treated. Buserelin was given daily (19·2 pmol/kg body wt); norethindrone was given orally three times / week (1·47 μmol/kg body wt). Bone resorption was monitored by measuring the urinary excretion of hydroxyproline and 45Ca and bone 45Ca content. Buserelin-treated rats developed similarly depressed plasma oestradiol-17β values in the presence and absence of progestogen, and both groups given buserelin had significantly smaller uteri than controls or rats given norethindrone without buserelin. However, norethindrone did not prevent buserelin-mediated increases in bone resorption. At the end of the study total body calcium values (mean ± s.d.) in the four groups were (mg) respectively; 2594 ± 123; 2260 ± 92 (P < 0·001 compared with controls); 2616 ± 221; 2415 ± 130 (P < 0·02 compared with controls). Rats given norethindrone and buserelin in combination had higher values (P < 0·02) than animals given buserelin alone, but significantly less total body calcium than controls (P < 0·02). We conclude that progestogen confers partial, but not complete, protection of the skeleton of buserelin-treated rats from oestrogen-deficiency bone loss.

Journal of Endocrinology (1993) 137, 27–33
Pulsatile prolactin secretory patterns throughout the oestrous cycle in the rat
Authors: Lafuente A. , Marcó J. , Esquifino A. I.
Published: December 2008

Prolactin secretion throughout the oestrous cycle in the rat remains at a low level and fairly constant, with the exception of the surge at pro-oestrus. The present study was designed to characterize possible changes in pulsatile patterns of prolactin during the oestrous cycle of the adult female rat. Mean values of prolactin increased from dioestrus-2 to pro-oestrus and then decreased to the values found at dioestrus-1. The number of peaks remained fairly constant in any phase of the oestrous cycle. The absolute amplitude of the peaks increased numerically but was not statistically significant from dioestrus-2 to pro-oestrus then decreasing until dioestrus-1. No changes in the relative amplitude or duration of the peaks throughout the oestrous cycle were detected. The results indicated that there is a similar pulsatile pattern of prolactin at any stage of the oestrous cycle, when samples were obtained during the morning.

Journal of Endocrinology (1993) 137, 43–47
Isolation of FSH from bovine pituitary glands
Authors: Wu J.-B. , Stanton P. G. , Robertson D. M. , Hearn M. T. W.
Published: December 2008

An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5·7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay.

SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19·5 kDa and 15·8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with α- and β-subunits, and a third minor (< 20%) sequence consistent with the α-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators.

Journal of Endocrinology (1993) 137, 59–68
Effects of FSH and testosterone on intratesticular insulin-like growth factor-I and specific germ cell populations in rats treated with gonadotrophin-releasing hormone antagonist
Authors: Spiteri-Grech J. , Weinbauer G. F. , Bolze P. , Chandolia R. K. , Bartlett J. M. S. , Nieschlag E.
Published: December 2008

Evidence derived predominantly from a series of in-vitro studies indicates that insulin-like growth factor-I (IGF-I) plays an important role as a paracrine and autocrine regulator within the testis. We investigated the effects of FSH substitution on spermatogenesis and testicular IGF-I content in rats treated with gonadotrophin-releasing hormone (GnRH) antagonist (ANT) and injected with ethane dimethane sulphonate (EDS), a Leydig cell toxin. FSH treatment partially prevented the marked decrease in intratesticular IGF-I in rats treated with only GnRH antagonist but not in GnRH antagonist + EDS-treated animals (controls, 191·0 ± 4·5; ANT, 80·1 ± 5·6; ANT + EDS, 81·6 ± 3·4; ANT + EDS + FSH, 86·3 ± 1·4; ANT + FSH, 137·7 + 7·3 (s.e.m.) ng/testis). Correlation analysis of testicular IGF-I content with the number of pachytene spermatocytes and round spermatids per cross-section revealed r values of 0·77 and 0·74 respectively (P < 0·001). We then analysed the same parameters in GnRH antagonist-treated rats which, in addition, received daily injections of the antiandrogen flutamide, in order to investigate the potential role of testosterone, as opposed to other Leydig cell products, in the regulation of spermatogenesis and testicular IGF-I. FSH treatment prevented regression of spermatogenesis in rats treated with GnRH antagonist alone but not in GnRH antagonist-and flutamide-treated rats. Testicular IGF-I content was altered in all treatment groups with the most marked changes observed in animals receiving GnRH antagonist and flutamide (FL) with or without FSH (5 or 10 IU/rat twice a day; FSH5 and FSH10) (ANT + FL, 150·32 ± 7·38; ANT + FL + FSH5, 165·28 ± 5·92; ANT + FL + FSH10, 160·17 ± 11·73 vs 464·51 ± 36·04 ng/testis for controls; P < 0·05). Those animals treated with GnRH antagonist and/or FSH had testicular IGF-I levels which were significantly lower than controls and higher than the three groups receiving flutamide and antagonist. A highly significant correlation was established between intratesticular IGF-I and the number of pachytene, round and elongated spermatids per cross-section (r = 0·8 for all three germ cell types). The data presented here provide direct in-vivo evidence for the importance of Leydig cell–Sertoli cell interactions in regulating testicular IGF-I content and spermatogenesis in the testis.

Journal of Endocrinology (1993) 137, 81–89
Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production
Authors: Yateman M. E. , Claffey D. C. , Cwyfan Hughes S. C. , Frost V. J. , Wass J. A. H. , Holly J. M. P.
Published: December 2008

Human dermal fibroblasts produce a number of insulin-like growth factor-binding proteins (IGFBPs) including the main circulating form, IGFBP-3. It has been suggested that the regulation of IGFBP secretion may play a major role in modulating insulin-like growth factor (IGF) bioactivity. We have quantified the effects of two cytokines, transforming growth factor-β1 (TGF-β1) and tumour necrosis factor-α (TNF-α) which have opposing actions on fibroblast IGFBP-3 production, and examined their subsequent role in IGF-I mitogenesis. TGF-β1 caused a dose-dependent increase in IGFBP-3 in serum-free fibroblast-conditioned media. TGF-β1 (1 μg/l) resulted in immunoreactive IGFBP-3 levels reaching 286·5 ± 22·4% of control after 20 h, the increase being confirmed by Western ligand blot. TNF-α caused a dose-dependent decrease in fibroblast IGFBP-3 secretion, 1 μg TNG-α/l reducing IGFBP-3 levels to 32·1 ± 11·% of control. This effect was not due to cytotoxicity and was not cell-density-dependent. Fibroblast proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric cytochemical bioassay. The addition of IGF-I resulted in dose-dependent growth stimulation after 48 h, the effective range being 20–100 μg/l. The IGF-I analogue Long-R3-IGF-I which has little affinity for the IGFBPs was approximately 20-fold more potent in this assay, and was unaffected by exogenous IGFBP-3. While the addition of 1 μg TGF-β1/l to increasing doses of IGF-I resulted in a fourfold decrease in mitogenic sensitivity to the IGF-I, no such effect was seen with Long-R3-IGF-I. Conversely, 1 μg TNF-α/l increased fibroblast IGF-I sensitivity five-fold, an effect not observed with the IGF-I analogue. Such data suggest that endogenous IGFBP-3 inhibits IGF-I bioactivity and that the regulation of IGF mitogenesis by TGF-β1 and TNF-α can occur via local IGFBP modulation. This may represent a mechanism by which complex growth signals are co-ordinated in vivo.

Journal of Endocrinology (1993) 137, 151–159