bluuebunny/crossref_metadata_2025 huggingface dataset:
Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls
Authors: Breier B. H. , Gallaher B. W. , Gluckman P. D.
Published: December 2008

This report describes essential requirements for the validation of a radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) and presents solutions to some problems and pitfalls commonly observed. The preparation of IGF-I to be used as radioligand or standard has to be selected carefully since some IGF-I preparations are contaminated with variants which demonstrate different potencies for different antisera used in the RIA.

Accurate assessment of IGF-I levels in blood plasma requires an efficient extraction method for the IGF-binding proteins (IGFBPs). Extraction methods to remove the influence of IGFBPs in the RIA were compared using blood plasma of considerable differences in IGF-I/IGFBP ratios. Acidification of plasma before column chromatography on Sephadex G-75 (G75) is generally considered to be the most reliable extraction method, but it is very time-consuming. The acid–ethanol extraction (AE) of plasma is not valid in many situations. Non-parallel displacement to the IGF-I standard was observed with AE-extracted plasma samples in the RIA. In addition, a comparison of IGF-I values obtained in the RIA after AE or G75 extraction of fetal ovine plasma has shown no significant correlation.

We report an extraction technique based on a modified AE extraction followed by cryo-precipitation (AEC). AEC extraction on blood plasma reduced residual IGFBPs to a level that did not interfere in the assay. Furthermore, AEC-extracted plasma samples showed parallel displacement in the RIA to highly purified preparations of authentic IGF-I. We observed high correlations, with a slope close to unity, of IGF-I values obtained in the RIA using the AEC or G75 extraction for plasma from different species including adult and fetal sheep, rat, mouse and man. The AEC extraction provides a rapid and simple alternative to G75 extraction for blood plasma from a variety of species provided that high-affinity antisera are used for the RIA.

Journal of Endocrinology (1991) 128, 347–357
Differential effects of epidermal growth factor on the differentiation of type A spermatogonia in adult mouse cryptorchid testes in vitro
Authors: Haneji T. , Koide S. S. , Tajima Y. , Nishimune Y.
Published: December 2008

The effect of epidermal growth factor (EGF) on testicular germ cell differentiation was investigated. Testicular fragments from surgically prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of EGF. Histological sections of testis were examined under a light microscope and each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. EGF at concentrations ranging from 100 to 200 ng/ml induced differentiation of type A spermatogonia. The observed maximal stimulatory activity of EGF at a concentration of 100 ng/ml was 30% of the positive control cultures treated with calf serum. EGF at concentrations ranging from 1 to 100 ng/ml significantly inhibited the mitotic activity of FSH, FSH plus retinol, or FSH plus fetuin on type A spermatogonia and their differentiation. The number of type A spermatogonia in testes cultured with FSH, FSH plus retinol, or FSH plus fetuin decreased when EGF was added. On the other hand, EGF stimulated the differentiation of type A spermatogonia induced with fetuin but did not influence retinol-induced differentiation. It is proposed that EGF inhibits testicular germ cell differentiation by blocking the proliferation of type A spermatogonia stimulated by FSH.

Journal of Endocrinology (1991) 128, 383–388
Effects of bovine follicular fluid on the secretion of LH and FSH in inhibin-immunized seasonally anoestrous ewes
Authors: Knight P. G. , Wrathall J. H. M. , Glencross R. G. , McLeod B. J.
Published: December 2008

It has been shown previously that treatment of seasonally anoestrous ewes with steroid-free bovine follicular fluid (FF), a crude inhibin-containing preparation, leads to a decrease in plasma FSH level which is accompanied by a marked increase in pulsatile LH secretion. Since FF contains several factors (e.g. activin, follistatin, unidentified components) other than inhibin, which might act to modify gonadotrophin secretion, it was of interest to establish whether these concurrent effects of FF on FSH and LH secretion persisted in ewes which had been actively immunized against a synthetic peptide replica of the α subunit of bovine inhibin. In June 1989 (anoestrous period) groups of inhibin-immune and control ewes (n = 5 per group) received 6-hourly s.c. injections of either bovine serum (2 ml) or one of two doses of FF (0·5 ml or 2 ml) for 3 days. Blood was withdrawn at 6-h intervals for 6 days beginning 24 h before the first injection. On the final day of treatment, additional blood samples were withdrawn at 15-min intervals for 8 h to monitor pulsatile LH secretion. Ewes were then challenged with exogenous gonadotrophin-releasing hormone (GnRH; 2 μg i.v. bolus) to assess pituitary responsiveness. In control ewes, FF promoted a dose-dependent suppression of basal (maximum suppression 65%; P < 0·01) and post-GnRH (maximum suppression 72%; P < 0·01) levels of FSH in plasma. This was accompanied by an increase (P < 0·01) in LH pulse frequency from 1·40±0·24 (s.e.m.) to 3·20±0·37 pulses/8 h. In contrast, FF did not affect secretion of either FSH or LH in inhibin-immunized ewes. However, mean plasma LH levels in immunized ewes were significantly lower (43%; P < 0·02) than in control ewes, irrespective of treatment. These findings indicate that in the anoestrous ewe the ability of FF to suppress plasma FSH is due entirely to its content of inhibin, that FF-induced enhancement of pulsatile LH secretion is mediated by inhibin, rather than some additional component of FF, and that immunoneutralization of endogenous inhibin can reduce LH secretion.

Journal of Endocrinology (1991) 128, 403–410
The effect of genetic selection for milk yield on the response to growth hormone secretagogues in immature cattle
Authors: Løvendahl P. , Angus K. D. , Woolliams J. A.
Published: December 2008

Eighty 4-month-old calves of both sexes and of two selected lines differing by 70 kg in their predicted total yield of milk fat and protein were injected intravenously with three of four GH secretagogues: these were, per kg liveweight; (i) 0·2 μg human GH-releasing factor(1–29) (GRF), (ii) 0·2 μg TRH, (iii) a combination of (i) and (ii), and (iv) 0·1 g arginine hydrochloride. The response of GH was measured for 2 h following administration. Geometric mean concentration of the 5-, 10-, 15- and 20-min samples following GRF, TRH and their combination were 29·3, 19·5 and 156 μg/l compared with baseline means of 6·5, 10·0 and 12·6 μg/l respectively, and for arginine (in which the mean response included the 30-min instead of the 5-min sample) 14·6 μg/l compared with a baseline of 8·31 μg/l. The line selected for greater yield responded more to each secretagogue by 1·53-fold following GRF (P < 0·01), 1·34-fold following TRH (P < 0·05), 1·11-fold following the combination (P > 0·01) and 1·26-fold following arginine (P < 0·1). Females responded 2·3-fold more than males following GRF administration (P < 0·001), only 1·2-fold more following TRH (P > 0·1), but less (0·63-fold) than males when GRF was combined with TRH (P < 0·05). For all secretagogues the concentration of GH before administration was important in determining the size of response (P < 0·001). It was concluded that the increased release of GH following the administration of GRF and TRH was a direct result of selection for dairy merit and that increased yields during lactation may, in part, be mediated directly through pituitary responsiveness.

Journal of Endocrinology (1991) 128, 419–424
Cholecalciferol has no effect on calcium and inorganic phosphorus balance in a naturally cholecalciferol-deplete subterranean mammal, the naked mole rat (Heterocephalus glaber)
Authors: Buffenstein R. , Yahav S.
Published: December 2008

Naked mole rats, Heterocephalus glaber, have no obvious source of cholecalciferol (D3) available to them, given their underground habitat and tubiferous diet. They have undetectable levels of 25-OH-D3 and as such appear to be naturally deplete in D3. The effect of an oral D3 supplement on mineral balance and homeostasis was therefore investigated.

This D3 treatment did not affect circulating levels of Ca2+ and inorganic phosphorus (Pi). Nor did D3 treatment affect mineral intake and absorption. The Ca2+ and Pi present in the food was efficiently extracted and absorbed, resulting in an apparent fractional absorption (AFA) efficiency exceeding 98%. Irrespective of D3 treatment, the amount of Ca2+ and Pi absorbed was positively correlated with the amount ingested, suggesting that intestinal uptake is by a passive D3-independent process. After D3 supplementation urinary Ca2+ secretion was unchanged; however, the amount of Pi excreted in the urine increased (P ≤ 0·05). This resulted in a concomitant decline in Pi AFR (P ≤ 0·02 from 99·95±0·02% to 99·82±0·03%). Almost all the Ca2+ and Pi in the glomerular filtrate were reabsorbed, facilitating AFR efficiencies that approach physiological maxima (> 99%). Changes in AFR efficiency with D3 supplementation are therefore of no biological significance. Net mineral flux of both elements, irrespective of D3 treatment, was positive. It is speculated that the ever-growing incisors of these animals act as mineral dumps and assist in the tight regulation of plasma Ca2+ and Pi.

These data suggest that naked mole rats utilize mechanisms independent of D3 in regulating mineral homeostasis and are therefore well-adapted to an environment devoid of sunlight.

Journal of Endocrinology (1991) 129, 21–26
Effects of truncated glucagon-like peptide-1 on the responses of starved sheep to glucose
Authors: Faulkner A. , Pollock H. T.
Published: December 2008

The effects of i.v. glucagon-like peptide-1-(7–36)amide (GLP-1; 10 μg) on starved sheep given an i.v. glucose load (5 g) were studied. Plasma insulin concentrations rose significantly more after glucose administration in fed than in starved sheep. Giving GLP-1 to starved sheep increased the insulin response to the glucose load. The rise in plasma insulin concentrations in starved sheep given GLP-1 was similar to that observed in fed sheep. Plasma glucose concentrations returned to normal values more quickly in the starved sheep given GLP-1 than in starved sheep not given gut hormone. Plasma concentrations of free fatty acid, urea and α-amino nitrogen decreased more quickly following glucose administration in starved sheep given GLP-1 than in those not given GLP-1. The data suggest a role for GLP-1 in regulating plasma insulin concentrations and hence metabolism in ruminant animals. The possible role of gut hormones in ruminants is discussed.

Journal of Endocrinology (1991) 129, 55–58
Immunohistochemical and in-situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland
Authors: Takeda H. , Chang C.
Published: December 2008

Ontogeny of the androgen receptor in the mouse prostate during fetal and post-natal periods was re-evaluated by histochemistry using a monoclonal antibody and by in-situ hybridization using a cDNA probe. Unlike steroid autoradiographic techniques which show ligand-binding sites in autoradiographs, these two techniques make it possible to investigate the expression of receptor protein and the transcription of its mRNA directly at the cellular level. Our previous autoradiographic data have demonstrated that only the mesenchyme takes up androgen during the fetal period; no incorporation of androgen by the epithelium has been observed in the fetus, although this does occur in later development. This observation indicated that the morphogenesis of prostatic epithelium is induced by the androgen-activated surrounding mesenchyme. In the post-natal prostate, the present data are nearly the same as those obtained by autoradiographic techniques; ligand-binding activity and expression of the receptor and its mRNA are much higher in the epithelium than in the stroma. On the other hand, the present study indicated that the fetal epithelium, which had shown no ligand-binding activity by autoradiographic analysis, expressed the same amount of receptor protein and its mRNA as the fetal mesenchyme. The results suggest that androgen receptor protein is expressed in the fetal epithelium but may not be able to bind to androgens until post-natal development.

Journal of Endocrinology (1991) 129, 83–89
Effects of corticosterone on the secretion of corticotrophin-releasing factor, arginine vasopressin and oxytocin into hypophysial portal blood in long-term hypophysectomized rats
Authors: Sheward W. J. , Fink G.
Published: December 2008

To investigate the feedback effects of corticosterone on the secretion of corticotrophin-releasing factor-41 (CRF-41), oxytocin and arginine vasopressin (AVP), hypophysial portal vessel blood was collected from control (intact) and long-term (6–8 weeks) hypophysectomized rats. In preliminary experiments in rats anaesthetized with urethane, long-term hypophysectomy resulted in a significant increase in the secretion of oxytocin and AVP; the hypothalamic contents of oxytocin and AVP were also increased in comparison with pituitary-intact rats. In long-term hypophysectomized rats anaesthetized with sodium pentobarbitone, but not with urethane, the output of CRF-41 into portal blood was increased twofold in comparison with that in control rats. In long-term hypophysectomized rats anaesthetized with pentobarbitone, the i.v. infusion of corticosterone (7·2 nmol/min) for a 2 h period of portal blood collection did not alter the secretion of CRF-41, oxytocin or AVP into portal blood; however, the secretion of CRF-41 and, to a lesser extent, AVP was significantly reduced in hypophysectomized rats by continuous corticosterone replacement, by a pellet of corticosterone implanted s.c. for 5 days before portal blood collection. These results confirm that the secretion of CRF-41 is differently affected by the anaesthetics urethane and pentobarbitone, and in long-term hypophysectomized rats show (i) that there were no apparent feedback effects of corticosterone infusion over a 2 h period on the secretion of any of the peptides studied, (ii) that late delayed feedback effects of continuous administration of corticosterone are mediated by a reduction in CRF-41 and AVP output, and (iii) that corticosterone has no effects on oxytocin secretion into portal blood.

Journal of Endocrinology (1991) 129, 91–98
Regional differences in catecholamine concentrations in bovine ovaries analysed by high-performance liquid chromatography
Authors: Denning-Kendall P. A. , Wild M. L. , D. C. Wathes
Published: December 2008

Bovine corpora lutea and ovarian stroma were analysed by high-performance liquid chromatography for catecholamine content. High concentrations (up to 102 nmol/g wet weight) were found in both 'central' stroma, containing many blood vessels, and 'peripheral' stroma. Central stroma contained noradrenaline and some dopamine, whereas peripheral stroma contained a higher proportion of dopamine and also significant amounts of 3,4-dihydroxyphenylacetic acid (DOPAC). Occasional samples of stroma had very high amounts of dopamine, suggesting that it is stored in specific regions. Corpora lutea, although devoid of direct innervation, contained dopamine (up to 5·3 nmol/g) and noradrenaline (up to 1·2 nmol/g). The average dopamine: noradrenaline molar ratio was 1·19 : 1 and the concentrations of dopamine and noradrenaline were highly correlated (P < 0·002). The concentration of dopamine was significantly higher in the early luteal phase of the oestrous cycle than during the rest of the cycle or in pregnancy. The levels of noradrenaline and dopamine present in corpora lutea are sufficient to modulate the production of both oxytocin and progesterone by luteal cells in vitro.

Journal of Endocrinology (1991) 129, 221–226
Androgen receptor in the Harderian gland of Rana esculenta
Authors: d'Istria M. , Chieffi-Baccari G. , Di Matteo L. , Minucci S. , Varriale B. , Chieffi G.
Published: December 2008

An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1·9±1·3 (s.d.) nmol/l (n = 26) for the cytosolic extract and Kd = 0·9±0·8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5α-dihydrotestosterone); oestradiol-17β showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17β was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August.

The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes.

Journal of Endocrinology (1991) 129, 227–232
Thyrotrophin, but not forskolin, increases intracellular free calcium and calmodulin in pig thyroid cells
Authors: Ye Q. L. , Wagner M. , Smythe S. , Neil S. Mac
Published: December 2008

The effects of TSH and forskolin were examined on intracellular free calcium ([Ca2+]i) and calmodulin in normal pig thyroid cells in culture. TSH was found to produce acute increases in [Ca2+]i in pig cells. Responses were seen at concentrations of TSH between 0·01 and 10 mU/ml. Sensitivity to TSH was greater in adherent monolayers of cells than in cell suspensions and was also greater in subconfluent rather than confluent monolayers of cells. The increase in [Ca2+]i in response to TSH represented just over a doubling in [Ca2+]i whether examined at 22 °C or 37 °C. Forskolin failed to affect [Ca2+]i. TSH increased [Ca2+]i in the absence of extracellular calcium.

TSH, but not forskolin, produced a significant increase in intracellular calmodulin after 3 days of culture of cells with TSH. The increase in calmodulin was of the order of 60% and did not relate to any effect of TSH on thyroid cell number.

Journal of Endocrinology (1991) 129, 291–299
Involvement of the adrenergic system on the release of prolactin and lactogenesis at the end of pregnancy in the rat
Authors: Jahn G. A. , Deis R. P.
Published: December 2008

The part played by the adrenergic system on the release of prolactin and lactogenesis induced by prostaglandin F2α and the antiprogesterone RU 486 was studied in pregnant rats. Two doses of prostaglandin F2α (150 μg) administered at 08.00 and 12.00 h on day 19 of pregnancy induced, at 12.00 h on day 20 (24 h after administration), a significant increase in the serum concentration of prolactin, with a significant decrease in serum progesterone levels. These hormonal changes significantly augmented casein and lactose levels in the mammary gland. Treatment with RU 486 (2 mg/kg) at 08.00 h on day 19 augmented casein and lactose concentrations in the mammary gland at 12.00 h on day 20 without modifying serum concentrations of prolactin and progesterone. The adrenergic antagonists, propranolol (3 mg/kg), metoprolol (10 mg/kg), ICI 118 551 (200 μg/kg), idazoxan (100 μg/kg) and prazosin (10 mg/kg), were administered s.c. at 12.00 and 20.00 h on day 19 and 08.00 h on day 20 of pregnancy to intact rats or to rats previously treated with RU 486 or prostaglandin F2α. These adrenergic antagonists did not modify serum prolactin or progesterone levels in intact or RU 486-treated rats, but serum prolactin levels in the prostaglandin F2α-treated group were significantly reduced by treatment with propranolol, metoprolol or prazosin. In addition, propranolol and ICI 118 551 also decreased the casein and lactose concentrations in the mammary glands of RU 486- and prostaglandin F2α-treated rats, while the other compounds had no effect. We also studied the effect of adrenergic antagonists on the release of prolactin and lactogenesis induced by the physiological decrease in progesterone at the end of pregnancy. On day 21 of pregnancy at 18.00 h, serum progesterone levels in intact rats were lower than 40 nmol/l, while serum prolactin and casein and lactose concentrations in the mammary gland were higher compared with values measured at 12.00 h on day 20. Treatments with propranolol, metoprolol or prazosin administered at 20.00 h on day 20 and 08.00 and 14.00 h on day 21 of pregnancy were capable of significantly reducing serum prolactin concentrations while only propranolol decreased mammary casein and lactose. The effect of propranolol was not mediated through a reduction in serum placental lactogen measured by Nb2 lymphoma cell bioassay.

These results show that the adrenergic system participates, through α1 and β1 receptors, in the regulation of prolactin release induced by the decrease in progesterone in pregnant rats. They also show that β2-adrenergic receptors play a role in the induction of casein and lactose synthesis in the mammary gland.

Journal of Endocrinology (1991) 129, 343–350
Kinetic evidence for two components in the priming effect of LH-releasing hormone in the rat
Authors: Johnson M. S. , Mitchell R.
Published: December 2008

The priming effect of LHRH on LH release from prooestrous rat hemipituitary glands in vitro was analysed by kinetic approaches. Concentration–response curves for LHRH-, K+- and ionomycin-induced LH release were constructed for initial exposure to the secretagogues and after 'priming' with a low dose of LHRH (100 pg/ml). These data were analysed by a non-linear curve-fitting programme to reveal the potency and maxima of the responses before and after priming. The parameters obtained from the curves fitted to the LHRH concentration–response curves showed that two changes had occurred as a result of priming. There was an increase in the maximum amount of hormone released and also a relatively greater ability for low concentrations of LHRH to cause release (increased potency). The data for K+ and ionomycin revealed only one change as a result of priming, an increase in the maximum amount of hormone available for release.

The data indicate that LHRH, after self-priming, releases more hormone by at least two routes, one represented by a general increase in stimulus–secretion coupling (which is available to K+ and ionomycin), the other a specific up-regulation of signal transduction by the LHRH receptor–effector system.

Journal of Endocrinology (1991) 129, 351–355
Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium
Authors: Bernal A. López , Buckley S. , Rees C. M. P. , Marshall J. M.
Published: December 2008

The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 ([3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1·5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 μmol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (≥ 100 μmol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 μmol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1–10 μmol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation.

Journal of Endocrinology (1991) 129, 439–445
Metabolic clearance rate of testosterone in male epileptic patients on anti-convulsant therapy
Authors: Wheeler M. J. , Toone B. K. , Dannatt A. , Fenwick P. B. C. , Brown S.
Published: December 2008

There are several reports which state that male epileptics on anti-convulsant therapy have reduced sexual activity. We and others have shown that, although total testosterone is raised, the free testosterone concentration is reduced in this patient population. This could be a result of an increased metabolic clearance rate (MCR) of testosterone, inadequate secretion of LH to stimulate testosterone synthesis or inappropriately low testosterone production by the Leydig cells. We have examined these possibilities by measuring the MCR of testosterone in 15 male epileptics on anti-convulsant therapy.

In this group of patients, the mean LH (9·3±5·9 IU/l) and sex-hormone binding globulin (SHBG) (54·5±22·9 nmol/l) concentrations were significantly greater than those of five normal control subjects (4·7±1·11 IU/l and 26·0 ±7·0 nmol/l respectively). Mean total testosterone concentrations of the two groups were not significantly different but the mean percentage of free testosterone and free testosterone concentration were significantly lower in the patient population (2·06±0·43 vs 2·98±0·27 and 0·56±1·1 vs 0·79±0·7 pmol/l). The MCR of testosterone was significantly lower in the patients (773±322 vs 1354±443 1/day) and showed a positive correlation with the percentage of free testosterone. Therefore, our results suggest that the lowered free testosterone in male epileptics on anti-convulsant therapy is not due to an increased MCR of testosterone. The increased LH concentration suggests primary hypogonadism. This, in turn, could be responsible for low free testosterone levels in the presence of normal testosterone.

Journal of Endocrinology (1991) 129, 465–468
Influence of protein nutrition on the response of growing lambs to exogenous bovine growth hormone
Authors: MacRae J. C. , Bruce L. A. , Hovell F. D. DeB. , Hart I. C. , Inkster J. , Walker A. , Atkinson T.
Published: December 2008

Interactions between protein supply and the anabolic response to exogenous bovine (b) GH have been examined in two experiments using 28–35 kg lambs sustained entirely by intragastric infusion of volatile fatty acids (700 kJ/kg W0·75 per day) into the rumen and the casein (600 mg (low protein; LP) or 1200 mg (high protein; HP)/kg W0·75 per day) into the abomasum. Sheep received continuous i.v. infusions of bGH for 6 days in experiment 1 and for 18 days in experiment 2. Nitrogen balances were determined daily throughout both experiments and blood samples, from indwelling catheters, were assayed for GH, insulin-like growth factor-I (IGF-I), insulin and glucose.

Infusion of bGH increased plasma GH concentration by five- to sixfold in all animals. There was an increase in N retention in both HP and LP animals over the first 2–3 days of GH administration. HP animals sustained higher N retentions (31%; P < 0·05) throughout the GH administration but LP animals did not. In contrast, plasma IGF-I concentrations increased progressively over the first 72 to 96 h of GH administration in all sheep and thereafter remained significantly (P < 0·05) elevated until termination of the GH infusion. In lambs which received both HP and LP infusions in experiment 1 the increase in IGF-I concentration by day 6 of GH administration was significantly (P < 0·05) greater when they received the higher protein intake. Plasma insulin concentrations increased rapidly (P < 0·05) with the onset of GH administration to levels which were 2·5 (LP)- and 4·8 (HP)-fold greater than those observed in the pre-and post-GH periods. Glucose concentration also increased during GH administration (P < 0·05), by 35% in LP animals and by 58% in HP animals.

High protein availability appeared necessary to sustain a protein anabolic response where lambs received exogenous GH infusions, even though plasma IGF-I concentrations were elevated on both high and low protein treatments.

Journal of Endocrinology (1991) 130, 53–61
Studies on the testicular source of inhibin and its route of secretion in rams: failure of the Leydig cell to secrete inhibin in response to a human chorionic gonadotrophin/LH stimulus
Authors: Tilbrook A. J. , de Kretser D. M. , Clarke I. J.
Published: December 2008

To determine whether Leydig cells produce inhibin in the ram, Leydig cells were stimulated by administering human chorionic gonadotrophin (hCG) or raising the levels of endogenous LH by an injection of gonadotrophin releasing hormone (GnRH). Plasma concentrations of testosterone increased in the 72 h after either a single injection (P < 0·05) or two injections (P < 0·01) of hCG. Plasma concentrations of inhibin were not significantly influenced by either one or two injections of hCG. Administration of GnRH (1 μg) caused an 11-fold increase in plasma concentrations of LH but did not influence concentrations of inhibin in either the jugular or testicular veins (pampiniform plexus). In contrast, concentrations of testosterone were increased by about fourfold in both jugular (P < 0·01) and testicular (P < 0·05) veins. The concentrations of inhibin in the testicular vein were 1·3-fold higher than in the peripheral plasma (P < 0·05) both before and following treatment with GnRH whereas the concentrations of testosterone were 18- to 21-fold greater than in peripheral concentrations.

In view of the difference in concentrations of inhibin between testicular and jugular veins, in a further experiment a sample was taken from the jugular vein, a vein located in the tunica vasculosa of the testis (testicular vein) and from a vein (spermatic vein) and lymph vessels located in the spermatic cord. The mean (± s.e.m.) concentrations of inhibin were highest in the testicular lymph (45·93±4·21 μg/l; P < 0·001) compared with the peripheral (4·14±0·31 μg/l), spermatic (8·0±1·17 μg/l) or testicular (6·4±0·82 μg/l) plasma. Plasma concentrations of inhibin were significantly higher in the spermatic vein than in the testicular vein (P < 0·05) and jugular vein (P < 0·01), and concentrations of inhibin in the testicular vein were significantly (P < 0·05) higher than in the jugular vein. There were no significant differences in the concentrations of testosterone in the spermatic vein, testicular vein or testicular lymph but the concentrations of testosterone in the peripheral plasma were significantly (P < 0·05) less than in the testicular plasma or lymph.

These results suggest that, in the ram, the Leydig cell does not respond to hCG or endogenous LH by secreting inhibin or by influencing other cells within the testis to secrete inhibin within the time-frame of these experiments. The low testicular to jugular differences in the concentration of inhibin and the high concentrations of inhibin in the testicular lymph suggest that the lymph may be an important route of secretion of inhibin from the testis in the ram.

Journal of Endocrinology (1991) 130, 107–114
Effect of oestradiol benzoate and tamoxifen on the growth of and induction of progesterone receptors in the uterus and mammary gland of immature pigs
Authors: Lin C. L. , Buttle H. L.
Published: December 2008

Intramuscular injection of oestradiol benzoate (0·1, 1 or 10 μg/kg per day) and tamoxifen (0·1 or 1 mg/kg per day) to 6-week-old immature pigs for 7 days induced a dose-dependent increase in the wet weight of the uterus and in the total content of uterine DNA, RNA and protein. Both compounds also stimulated a dose-dependent increase in the concentration of progesterone receptors in uterine cytosolic extracts (in terms of either fmol/mg DNA or fmol/g uterus). The concurrent administration of tamoxifen with oestradiol benzoate provoked significant (P < 0·05) increases in total uterine protein and in the concentration of progesterone receptors (P < 0·01) compared with treatment with oestradiol benzoate alone. Hence tamoxifen is an oestrogen agonist in the uterus of immature pigs.

The effects of oestradiol benzoate and tamoxifen on mammary growth in immature pigs were assessed by image analysis of mammary sections across the gland (in a ventro-dorsal direction through the teat). Oestradiol benzoate at 10 μg/kg per day stimulated a fourfold increase in mammary duct area (P < 0·01), and tamoxifen, at doses of 0·1 or 1 mg/kg per day, stimulated a threefold increase (P < 0·05). Tamoxifen partially inhibited (P < 0·05) the effect of oestradiol benzoate. The concentration of progesterone receptors was found to be very heterogeneous in cytosol extracts of mammary tissue of immature pigs and independent of treatment with oestradiol benzoate and/or tamoxifen.

Journal of Endocrinology (1991) 130, 259–265
Serotoninergic stimulation of aldosterone secretion in the rat in vivo: role of the renin-angiotensin system
Authors: Davies E. , Rossiter S. , Edwards C. R. W. , Williams B. C.
Published: December 2008

Serotoninergic control of aldosterone secretion in vivo was investigated in conscious rats with indwelling arterial cannulae. Serial blood samples were taken from the animals before and after i.p. administration of 1 ml (4 g/l) 5-hydroxytryptophan (5-HTP), the precursor of serotonin, or saline and they were analysed for 5-HTP, serotonin, 5-hydroxyindoleacetic acid, plasma renin activity (PRA), corticosterone, aldosterone, sodium and potassium concentrations. The role of the renin-angiotensin system was investigated in animals pretreated for 1 week with the angiotensin-converting enzyme inhibitor captopril (25 mg/day). 5-HTP caused a significant increase in all parameters within 45 min except for sodium and potassium. Saline administration showed no significant effect. Captopril pretreatment did not impair the increase in any parameter by 5-HTP, with the exception of the aldosterone response which was significantly attenuated, though not completely.

The results show that administration of 5-HTP, which increases serum serotonin levels, stimulates PRA, aldosterone and corticosterone secretion. Captopril pretreatment inhibits the aldosterone response, suggesting that the aldosterone stimulatory properties of 5-HTP require the presence of angiotensin II, although it is unclear whether it acts in a mediatory or permissive capacity. The failure of captopril to inhibit the aldosterone response completely suggests the involvement of other mechanisms such as the hypothalamo-pituitary adrenal axis or a direct action of serotonin on the adrenal.

Journal of Endocrinology (1991) 130, 347–355
The use of low doses of ACTH in the investigation of adrenal function in man
Authors: Crowley S. , Hindmarsh P. C. , Holownia P. , Honour J. W. , Brook C. G. D.
Published: December 2008

We have investigated the adrenal response, in eight healthy adult men, to low doses of ACTH(1–24) in order to define a dose which will elicit a response similar to that obtained with the short Synacthen test. The studies were performed at 14.00 h and blood samples were withdrawn at 5-min intervals after an i.v. bolus injection of ACTH(1–24). The sampling interval was crucial in determining the timing of the peak response. Using sampling times of 0, 10, 15, 20, 25 and 30 min ensured detection of 47 out of 48 peaks. A dose-dependent rise in plasma cortisol concentration was observed with bolus injections of ACTH(1–24) between 30 and 250 ng/1·73 m2 body surface area. Increasing the dose to 500 ng/1 ·73 m2 (500 times less than that used in the short Synacthen test) elicited an increment of plasma cortisol concentration of 200 nmol/l or greater in all subjects. Pretreatment with dexamethasone (1 mg) did not alter the timing of the peak cortisol concentration but blunted the increase (pretreatment: median, 159 nmol/l; range 83–239; on dexamethasone; median 62 nmol/l; range 21–207; P = 0·04). These data suggest that a dose of ACTH(1–24) of 500 ng/1·73 m2 satisfies the criteria of the short Synacthen test and may provide a useful method of investigating adrenal function.

Journal of Endocrinology (1991) 130, 475–479
The purification and development of a radioimmunoassay for β-core fragment of human chorionic gonadotrophin in urine: application as a marker of gynaecological cancer in premenopausal and postmenopausal women
Authors: Lee C. L. , Iles R. K. , Shepherd J. H. , Hudson C. N. , Chard T.
Published: December 2008

The β-core fragment of human chorionic gonadotrophin (hCG) is a major part of the immunoreactive hCG-like material found in the urine of normal pregnant women. Patients with non-trophoblastic gynaecological malignancies have been found to have raised levels of urinary β-core. We describe the purification of β-core, the preparation of a polyclonal sheep antiserum and the development of radioimmunoassay. The minimum detection limit of this assay was 0·025 μg β-core/l. There was no significant cross-reaction with the free α-subunit, hLH, hFSH and hTSH (<0·7%), and only partial cross-reaction with intact hCG and free β-subunit of hCG (6·9 and 18%). Within-assay variability ranged from 2·03 to 12·5% and between-assay variability from 2·25 to 13·4%. The assay was applied to urine samples from 92 normal non-pregnant premenopausal women, 54 normal postmenopausal women and 65 women with active gynaecological disease (47 postmenopausal and 18 premenopausal). In normal premenopausal women the values ranged from < 0·025 to 0·62 μg β-core/l (median 0·043 μg β-core/l). The values for normal postmenopausal women ranged from <0·025 to 0·64 μg β-core/l (median 0·26 μg β-core/l). Postmenopausal women with gynaecological malignancy had values which ranged from <0·025 to 4·0 μg/β-core/l (median 0·31 μg β-core/l); premenopausal women in this group had values which ranged from < 0·025 to 1·15 μg β-core/l (median 0·12 μg β-core/l). On molecular sieve chromatography, the material found in the urine of normal postmenopausal women showed the physicochemical characteristics of authentic β-core. However, the only detectable serum gonadotrophin was LH. Considering the homology and evolution of hCG from LH, postmenopausal β-core-like material could be derived from the degradation of β-LH resulting in a fragment with essentially identical physicochemical and immunological properties. The use of β-core assay as a tumour marker must take account of the fact that this molecule occurs in the urine of normal subjects, and that the levels are substantially raised in normal postmenopausal women.

Journal of Endocrinology (1991) 130, 481–489
Pulsatile LH secretion in streptozotocin-induced diabetes in the rat
Authors: Dong Q. , Lazarus R. M. , Wong L. S. , Vellios M. , Handelsman D. J.
Published: December 2008

This study aimed to determine the effect of streptozotocin (STZ)-induced diabetes on pulsatile LH secretion in the mature male rat. LH pulse frequency was reduced by 56% and pulse amplitude by 54%, with a consequential decrease of 72% in mean LH levels 8 days after i.v. administration of STZ (55 mg/kg) to castrated Wistar rats compared with castrated non-diabetic controls. Twice daily insulin treatment completely reversed all parameters of pulsatile LH secretion to control values. Food-restricted non-diabetic controls, studied to distinguish the metabolic effect of diabetes from that of concurrent weight loss, demonstrated a 34% reduction in LH pulse frequency but no significant changes in LH pulse amplitude or mean LH levels compared with non-diabetic controls given free access to food. To distinguish whether the decreased LH pulse amplitude in diabetes was due to a reduction in either the quantity of hypothalamic gonadotrophin-releasing hormone (GnRH) released per secretory episode or to decreased pituitary responsiveness to GnRH, the responsiveness of the pituitary to exogenous GnRH (1–1000 ng/kg body weight) was tested in diabetic rats after castration, using a full Latin square experimental design. The net LH response (total area under response curve over 40 min following GnRH) was decreased by 33% (P=0·001) in diabetic compared with control rats. The decreased LH pulse frequency in STZ-induced diabetes therefore suggests that the metabolic effect of diabetes is to decelerate directly the firing rate of the hypothalamic GnRH pulse generator independent of testicular feed-back. These effects were fully reversed by insulin treatment and were only partly due to the associated weight loss. The impaired pituitary responsiveness to GnRH is at least partly involved in the reduction of LH pulse amplitude.

Journal of Endocrinology (1991) 131, 49–55
The influence of vanadate on insulin counter-regulatory hormones in obese fa/fa rats
Authors: Brichard S. M. , Ongemba L. N. , Kolanowski J. , Henquin J. C.
Published: December 2008

Vanadate has been shown to improve glucose homoeostasis in mildly glucose-intolerant and severely insulin-resistant fa/fa rats. The present study examined whether changes in insulin counter-regulatory hormones contribute to this beneficial effect of vanadate. Since oral administration of Na3VO4 caused a decrease in food intake and stopped the increase in body weight, vanadate-treated fa/fa rats were compared with both controls with food available ad libitum and pair-fed rats. Slightly lower plasma glucose levels were maintained in conjunction with markedly lower plasma insulin levels in vanadatetreated rats, and this effect was not simply due to the smaller body weight of the animals. Compared with control rats, treatment with vanadate affected neither basal plasma glucagon levels nor the increase in glucagon levels observed after insulin-induced hypoglycaemia or after i.v. injection of arginine. Compared with pair-fed rats, treatment with vanadate prevented the fall in basal plasma glucagon and its exaggerated rise in response to insulin that mere food restriction produced. Plasma corticosterone levels were high in fa/fa rats. Vanadate and pair-feeding similarly decreased basal plasma levels of corticosterone as well as nocturnal corticosteronuria. Thus the attenuation of the hypercorticism of fa/fa rats results from the reduction in body weight gain rather than from a specific action of vanadate. Vanadate did not influence urinary excretion of noradrenaline, an index of neural sympathetic activity, but prevented the increase in adrenaline excretion, an index of adrenal medulla activity, that was produced by food restriction in pair-fed rats. In conclusion, vanadate administration has no or little specific effects on three major insulin counter-regulatory hormones. This reinforces the suggestion that the beneficial effects of vanadate on glucose homoeostasis in fa/fa rats are mainly due to a correction of insulin resistance in peripheral tissues.

Journal of Endocrinology (1991) 131, 185–191
Age-related changes of growth hormone secretory mechanisms in the rat pituitary gland
Authors: Parenti M. , Cocchi D. , Ceresoli G. , Marcozzi C. , Müller E. E.
Published: December 2008

The mechanisms underlying the age-related decrease and increase in somatotroph responsiveness to growth hormone-releasing factor (GHRF) and somatostatin respectively were studied in rat pituitary membranes in vitro.

Basal adenylate cyclase (AC) activity was similar in pituitary membranes from rats of 8 days (either sex) and male rats of 3 months, but it was almost threefold higher in membranes from male rats of 21–23 months. GHRF induced a lower percentage stimulation of AC activity in membranes from infant and old than adult rats. Somatostatin inhibited stimulation of AC induced by forskolin more effectively in membranes from adult than infant and old rats. In parallel experiments, since the tissue we used is formed by a mixed population of pituitary cells, we evaluated, for comparison, the effect on AC of neurohormones, i.e. vasoactive intestinal polypeptide (VIP) and dopamine which act primarily on lactotrophs. VIP induced a lower fold-stimulation of AC activity in membranes from infant and old than adult rats. Dopamine inhibited forskolin-induced stimulation of AC in the following rank order of magnitude: old, adult and infant rats, and was also more effective in inhibiting basal AC activity in old than in adult rats. The stimulatory and inhibitory G proteins (Gs and Gi) coupled to AC were measured indirectly by evaluating stimulatory and inhibitory effects of different concentrations of GTP on AC. GTP, at stimulatory concentrations, increased AC activity in membranes from infant and adult rats similarly whereas its effect was significantly greater in membranes from old rats. Conversely, GTP, at inhibitory concentrations, decreased AC activity similarly in membranes from adult and infant rats, whereas in old rats inhibition was apparent at more than a tenfold lower concentration of GTP.

These data suggest (1) that the greater somatotroph sensitivity to GHRF in terms of GH secretion of the early postnatal period is not due to supersensitive GHRF receptors but rather may be accounted for, at least partially, by the low function of somatostatinergic receptors; (2) that the inability of GHRF to stimulate GH release in aged rats probably results from an uncoupling between the GHRF receptor and the G protein; and (3) that in aged rats the decreased ability of somatostatin to inhibit AC activity, in spite of the high Gi activity, results from a reduced number of somatotroph cells and, hence, receptors.

Journal of Endocrinology (1991) 131, 251–257
Effects of β-endorphin and naloxone on corticosterone and cortisol release in the newt (Triturus carnifex): studies in vivo and in vitro
Authors: Zerani M. , Gobbetti A.
Published: December 2008

The effects of β-endorphin and its receptor antagonist, naloxone, on corticosterone and cortisol production in male and female Triturus carnifex were studied in vivo and in vitro. In the in-vivo experiment, the animals were injected s.c. with β-endorphin and/or naloxone, and killed after 15, 30, 90 and 360 min. In the in-vitro experiment, interrenal tissues, with and without added pituitary, were incubated with β-endorphin and/or naloxone for 15, 30, 60 and 120 min. The data obtained in vivo and in vitro from males and females were in agreement. Treatment with β-endorphin caused a significant decrease in corticoster-one and cortisol release, while naloxone induced an increase in the two corticosteroids at the same times as the decrease caused by β-endorphin. The combined β-endorphin plus naloxone treatment did not change corticosterone and cortisol levels. These results suggest that, in Triturus carnifex, opioids are involved in the regulation of the hypothalamo-pituitary-interrenal axis. In particular, the in-vitro results indicate a direct effect of opioids on interrenal steroidogenesis.

Journal of Endocrinology (1991) 131, 295–302