bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentrations of arachidonate metabolites, steroids and histamine in preovulatory horse follicles after administration of human chorionic gonadotrophin and the effect of intrafollicular injection of indomethacin
Authors: Watson E. D. , Sertich P. L.
Published: December 2008

This study investigated the sequence of hormonal changes within the preovulatory follicles of mares. Mares were injected i.v. with 2500 IU human chorionic gonadotrophin (hCG) when a preovulatory follicle of 35 mm in diameter was detected. Fluid was aspirated from preovulatory follicles before (0 h), and 12, 24 and 36 h after administration of hCG. Concentrations of progesterone, prostaglandin (PG) E2, PGF, 6-keto-PGF1α and thromboxane B2 in follicular fluid increased significantly (P<0·01) between 0 and 36 h. At 36 h, PGE2 was present in highest concentrations, followed by PGF and 6-keto-PGF1α; thromboxane B2 was present at lower concentrations than other prostanoids. Concentrations of 13,14-dihydro-15-keto-PGF2α increased significantly (P<0·05) between 24 and 36 h. Leukotriene B4, leukotriene C4 and histamine were present in follicular fluid at all sampling periods and did not change significantly over time.

In another experiment, buffered saline or indomethacin (either 100 or 500 μg) was injected into preovulatory follicles on the day that they reached 35 mm in diameter to determine whether blocking intrafollicular PG synthesis would affect ovulation. The interval between intrafollicular injection and ultrasonographic detection of luteinization was significantly longer (P<0·05) in mares treated with 500 μg indomethacin. Plasma progesterone concentrations were significantly (P<0·05) lower in indomethacin-treated mares than in control mares on the first 5 days after injection.

These results indicate that intrafollicular concentrations of PGs increase significantly before ovulation in mares and may be involved in the ovulatory process.

Journal of Endocrinology (1991) 129, 131–139
Binding sites for iodinated endothelin-1, endothelin-2 and endothelin-3 demonstrated on human uterine glandular epithelial cells by quantitative high-resolution autoradiography
Authors: Davenport A. P. , Cameron I. T. , Smith S. K. , Brown M. J.
Published: December 2008

Quantitative in-vitro receptor autoradiography has been used to localize and compare the anatomical distribution of binding sites for iodinated endothelins (ET-1, ET-2 and ET-3) in human uterus. Binding sites for the three iodinated isoforms had a similar gross anatomical distribution. The density of binding sites was significantly higher in the endometrium compared with the myometrium and greatest at the endometrial–myometrial junction.

In cross-competition experiments, unlabelled ET-1, ET-2, ET-3, sarafotoxin S6b and mouse vasoactive intestinal contractor (1 μmol/l) competed for the binding sites of all the iodinated peptides suggesting that ETs may bind to the same receptor. However, preproendothelin(110–130) (endothelin-like peptide) or preproendothelin(124–130) and other non-endothelin vasoactive peptides tested as a concentration of 1 μmol/l did not compete.

Micro-autoradiography revealed that high densities of iodinated ET-1, ET-2 and ET-3 binding sites were localized to glandular epithelial cells and blood vessels with lower levels in the myometrium and vascular smooth muscle, suggesting that these potent vasoactive and proliferative agents could play a role in the control of menstruation.

Journal of Endocrinology (1991) 129, 149–154
Regulation of differential release of α-melanocyte-stimulating hormone forms from the pituitary of a teleost fish, Oreochromis mossambicus
Authors: Lamers A. E. , Balm P. H. M. , Haenen H. E. M. G. , Jenks B. G. , Bonga S. E. Wendelaar
Published: December 2008

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of α-MSH (des-acetyl, mono-acetyl and di-acetyl α-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl α-MSH was, especially in comparison with monoacetyl α-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of α-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of α-MSH release, but did not modulate the relative abundance of the different forms of α-MSH released from the MSH cells. TRH was a potent stimulator of α-MSH release. It enhanced in vitro the release of di-acetyl α-MSH more than the release of mono-acetyl α-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Journal of Endocrinology (1991) 129, 179–187
Inherited primary hypothyroidism with thyrotrophin resistance in Japanese cats
Authors: Tanase H. , Kudo K. , Horikoshi H. , Mizushima H. , Okazaki T. , Ogata E.
Published: December 2008

Mutant cats were developed with non-goitrous primary hypothyroidism. They were clinically characterized by severely retarded growth, mild anaemia and high mortality in the young. They responded markedly to thyroid hormone replacement. Thyroid glands in the mutants were normal in position but slightly reduced in size. Laboratory studies revealed low serum concentrations of thyroxine (T4) and tri-iodothyronine (T3), and increased serum concentrations of TSH. Administration of TRH induced no further increase in TSH. Administration of exogenous TSH after suppression of endogenous TSH by T3 did not increase the serum concentration of T4 in the mutants, in sharp contrast with the threefold increase in serum T4 observed in the normal litter-mates. These findings suggest that the underlying pathogenesis of this disorder is unresponsive to TSH. Moreover, we found that the mutants were transmitted in an autosomal recessive manner.

Journal of Endocrinology (1991) 129, 245–251
Occurrence of sexual maturation in chronic opiate-treated female rats
Authors: MacDonald M. C. , Wilkinson M.
Published: December 2008

We have investigated the consequences of chronic morphine or fentanyl exposure on the timing of puberty in female Sprague–Dawley rats. The μ-receptor agonists morphine and fentanyl were either added to the drinking water or, in the case of fentanyl, in osmotic minipumps. Morphine and fentanyl treatment, beginning at postnatal day 22, delayed the time of the first ovulation/vaginal opening (VO). For example, morphine included in the drinking water (800 mg/l) delayed VO by 5 days. Fentanyl gave similar results but at lower concentrations (30 mg/l). Surprisingly, these treatments delayed but did not prevent VO; i.e. in the face of continued opiate treatment most rats ovulated normally. In contrast, identical doses of morphine blocked oestrous cyclicity in drug-naive adults rats. However, morphine-dependent rats, which have reached VO (and first ovulation) then became briefly acyclic before beginning regular cycles even though opiate treatment was continued. The effects of fentanyl on immature rats were identical with those of morphine, i.e. VO was delayed but ovulation occurred in spite of continued drug treatment. On the other hand, fentanyl did not prevent cyclicity subsequent to VO.

Our observation that immature female rats can reach first ovulation despite chronic opiate treatment suggests that some degree of tolerance to opiates may develop. Such a mechanism could operate in normal drug-free rats, via endogenous opioid peptides, in the timing of puberty.

Journal of Endocrinology (1991) 129, 253–259
Early glucocorticoid feedback in anterior pituitary corticotrophs: differential inhibition of hormone release induced by vasopressin and corticotrophin-releasing factor in vitro
Authors: Shipston M. J. , Antoni F. A.
Published: December 2008

Vasopressin and 41-residue corticotrophin-releasing factor (CRF-41) are physiological mediators of the hypothalamic control of pituitary ACTH secretion, whilst adrenocortical glucocorticoids are the major inhibitory factors regulating ACTH output. In the present study it was investigated in vitro whether the characteristics of early glucocorticoid inhibition of stimulated ACTH secretion would differ depending on the nature of the stimulus and the temporal relationship between secretagogue and steroid.

The experiments were carried out using perifused segments of rat adenohypophysis obtained from randomly cycling female rats. Repeated pulses (5 min) of CRF-41 or vasopressin were given at 1-h intervals for up to 7 h. The net release of ACTH became stable after the second secretagogue pulse. Administration of 0·1 μmol corticosterone/l 30 min before and during a 5-min pulse of 10 nmol CRF-41/l inhibited CRF-41-stimulated ACTH release to 60% of control. Stimulated hormone release remained suppressed at 90 min after the start of the corticosterone infusion and returned to control levels by 150 min. If corticosterone treatment (35 min total exposure) was started simultaneously with the CRF-41 pulse, no inhibitory effect of the steroid was observed at any subsequent time-point examined (60,90,120 and 150 min). In contrast, vasopressin-stimulated ACTH release was inhibited by approximately 50% when corticosterone was applied before, or simultaneously with, a 5-min pulse of 10 nmol vasopressin/l. The synthetic glucocorticoid type II receptor agonist RU28362, administered 30 min before and during a 5-min pulse of 10 nmol CRF-41/l, reduced CRF-41-stimulated ACTH release to 50% of control up to 2·5 h after the start of RU28362 application (although inhibition after 35 min exposure was not statistically significant). Inhibition of ACTH release stimulated by 10 nmol vasopressin/l was observed within 35 min of steroid application and was maintained up to 2·5 h after the initial application of RU28362. The action of RU28362 on CRF-41-stimulated ACTH release was blocked by inhibitors of transcription (actinomycin D) and translation (puromycin); notably these drugs did not modify the ACTH response to CRF-41. In contrast, actinomycin D as well as puromycin reduced vasopressin-stimulated ACTH release.

The data suggest that: (1) the timing of steroid application is important in determining the early glucocorticoid inhibition of CRF-41- but not vasopressin-stimulated ACTH secretion; (2) CRF-41 and vasopressin mobilize different pools of ACTH from the anterior pituitary gland; (3) type II glucocorticoid receptors and synthesis of new protein(s) are involved in the early inhibitory action of glucocorticoids; (4) depending on the timing and nature of the incident secretagogue, differential negative feedback inhibition of ACTH secretion may occur at the pituitary level in vivo.

Journal of Endocrinology (1991) 129, 261–268
Effect of hypophysectomy and acute administration of growth hormone (GH) on GH-receptor binding in chick liver membranes
Authors: Vanderpooten A. , Darras V. M. , Huybrechts L. M. , Rudas P. , Decuypere E. , Kühn E. R.
Published: December 2008

The effects of hypophysectomy on GH binding to liver membranes of young chicks were studied 3 days and 1 week after surgery. Specific binding of 125I-labelled chicken GH (cGH) to MgCl2-treated liver microsomal fractions of hypophysectomized animals was two- to fivefold greater than to those of sham-operated or control (non-operated) birds. This effect was due to a rise in binding capacity rather than a change in binding affinity of the GH receptor. Two daily injections of cGH (20 μg/animal) returned the number of hepatic GH receptors from hypophysectomized chicks to the level of the sham-operated ones. Administration of GH to the latter group did not cause a significant lowering of specific binding or number of receptors. No positive correlation between GH binding and plasma concentrations of insulin-like growth factor-I (IGF-I) was observed; although GH binding increased, IGF-I levels were lower for the hypophysectomized group. Since the number of hepatic GH receptors and the plasma GH levels were inversely correlated, it was concluded that the GH receptors in the liver of the chicken can be down-regulated by GH. This possibly explains why GH binding is low in posthatch and young chicks, because circulating GH concentrations are high during this period.

Journal of Endocrinology (1991) 129, 275–281
Circadian variation in the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male rats as analysed by flow cytometry
Authors: Carbajo-Pérez E. , Carbajo S. , Orfao A. , Vicente-Villardón J. L. , Vázquez R.
Published: December 2008

Flow cytometric analysis of nuclei stained with propidium iodide (PI) has been used to study the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male Sprague–Dawley rats at different times of the day. According to PI fluorescence intensity the relative numbers of cells in S phase (cells with a DNA content between that of somatic cells in interphase (2n) and that of somatic cells after duplication of the DNA prior to cell division (4n)) and G2/M phase (4n) were calculated. A significant circadian rhythm was found for cells in both the S phase (P < 0·05) and the G2/M phase (P < 0·01). The wave of cells in S phase with a peak at the middle of the light period (14.00 h) precedes by about 6 h the wave of cells in G2/M phase (peak at 20.00 h). Most of the DNA-replicating cells were found during the early S phase at 11.00 h, advancing further up to the middle of this phase at 14.00 h. Cells were distributed homogeneously throughout the S phase at 17.00 h. These data strongly suggest that the beginning of the light period triggers a wave of cells to leave G0/G1 into S phase.

Journal of Endocrinology (1991) 129, 329–333
The concentration of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in human umbilical cord serum at delivery: relation to fetal weight
Authors: Wang H. S. , Lim J. , English J. , Irvine L. , Chard T.
Published: December 2008

Serum levels of insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-1 (IGFBP-1) have been determined by radioimmunoassay in the maternal circulation (n = 91) and in the umbilical artery (n = 56) and vein (n = 90) of man. In both the umbilical artery and vein, the concentration of serum IGF-I showed an inverse correlation with birthweight (P < 0·005 and P < 0·001 respectively); the mean serum IGF-I levels in the small-for-gestational-age (SGA) group were significantly higher than those in average-for-gestational-age (AGA) neonates (P <0·01 and P < 0·001 respectively). However, maternal serum IGF-I showed no association with birthweight and there was no significant difference between the SGA and AGA groups. These observations imply that the production of IGF-I in the maternal and fetal compartments is independent and that there is unlikely to be transfer of IGF-I across the placenta. Serum IGFBP-1 levels in both maternal and umbilical cord blood (artery and vein) showed an inverse relation to birthweight (P <0·001, P<0·005 and P<0·001 respectively). Increased IGFBP-1 levels in the umbilical artery and vein were observed in the SGA group. These findings suggest that IGFBP-1 might inhibit the action of IGF-I in both the maternal and the fetal compartments and that the rise in IGFBP-1 could be a primary factor in retardation of fetal growth. Alternatively, circulating IGF-I and IGFBP-1 levels may only be a secondary reflection of local tissue events involved in fetal growth.

Journal of Endocrinology (1991) 129, 459–464
Stimulation of body weight gain of the mature female rat by bovine GH and bovine placental lactogen
Authors: Byatt J. C. , Staten N. R. , Schmuke J. J. , Buonomo F. C. , Galosy S. S. , Curran D. F. , Krivi G. G. , Collier R. J.
Published: December 2008

Mature female rats (200 g) were treated for 10 days with either recombinant bovine GH (bGH) or recombinant bovine placental lactogen (bPL) to compare the somatogenic responses elicited by these hormones. The treatments were administered by daily s.c. injection at four dose levels (0·19, 0·56, 1·67 and 5·0 mg/day). Both bGH and bPL stimulated significant increases in weight gain, but the slopes of the dose–response curves were different (P<0·05). Bovine PL was more potent than bGH (P<0·01) at the lowest dose, although there were no differences between treatment groups at the three higher doses. Feed consumption was stimulated more by bPL than bGH at all doses (P<0·001). The concentration of insulin-like growth factor-I (IGF-I) in blood plasma was increased by bGH in a dose-responsive manner and was higher than control at doses of 1·67 and 5 mg/day (P<0·05). Low doses of bPL stimulated increases in IGF-I similar to those with bGH. At the highest dose of bPL, however, there was no concomitant increase in plasma IGF-I. Nevertheless, the growth rate of the animals in this group matched that of the group given the highest dose of bGH. Receptor binding studies indicated that bPL bound to both GH and prolactin receptors. This is consistent with the growth data which suggest that bPL stimulated weight gain through a somatogenic mechanism as well as by another route, possibly mediated by lactogenic receptors.

Journal of Endocrinology (1991) 130, 11–19
Substance P stimulation of polyphosphoinositide hydrolysis in rat anterior pituitary membranes involves a GTP-dependent mechanism
Authors: Mau S. E. , Saermark T.
Published: December 2008

Substance P (SP) stimulates polyphosphoinositide breakdown in the rat anterior pituitary through an NK-1 receptor. In the present study we present evidence that the coupling between the SP–NK1 receptor complex and polyphosphoinositide-specific phospholipase C (PI-PLC) in rat anterior pituitary membranes may involve a mechanism consistent with a GTP-binding protein. The formation of inositol phosphates from [3H]myo-inositol-labelled anterior pituitary membranes induced by SP was potentiated by GTP and non-hydrolysable guanine nucleotides. The stimulatory effects of SP alone and SP plus GTP could be blocked by addition of GDP-β-S (guanosine 5-O-(thiodiphosphate)) in excess. Basal and SP plus guanine nucleotide-induced inositol phosphate formation were stimulated by fluoride, whereas the effect of SP alone was inhibited. Pretreatment of anterior pituitary membranes with sodium deoxycholate attenuated the inositol phosphate response elicited by GTP and GTP-γ-S, whereas basal and SP-stimulated inositol phosphate production showed a peak at 1 mg sodium deoxycholate/ml. SP, fluoride and guanine nucleotide stimulatory effects on hydrolysis of polyphosphoinositide (PPI) were unaffected by pretreatment of anterior pituitary cells with cholera or pertussis toxin for 12 h. Treatment of anterior pituitary membranes with cholera and pertussis toxin yielded [32P]ADP-ribosylation of two proteins with molecular masses of 45 and 41 kDa respectively.

We conclude that SP coupling to PI-PLC through the NK1 receptor in the rat anterior pituitary involves a GTP-binding mechanism distinct from the G-proteins associated with adenylate cyclase, Gs and Gi.

Journal of Endocrinology (1991) 130, 63–70
Effects of RU 486 on adrenal steroidogenesis in the guinea-pig
Authors: Provencher P. H. , Bélanger A , Fiet J.
Published: December 2008

Recent reports have shown that RU 486, a synthetic glucocorticoid and progestin antagonist, has direct effects on tissues secreting steroids. In order to characterize the effects of RU 486 on steroidogenesis further, guinea-pig fasciculata-glomerulosa (FG) cells in primary culture were treated for 48 h with RU 486. RU 486 caused an alteration of basal as well as ACTH-stimulated steroid secretion. Corticosterone and cortisol secretion decreased by 50% while the secretion of 17-hydroxyprogesterone and C19 steroids were increased. The activity of steroidogenic enzymes was measured using tritiated steroids. In RU 486-treated cells, the activity of 21-hydroxylase was dramatically inhibited while there was an increase in 17-hydroxylase and 17,20-desmolase activities. The effects of RU 486 on enzyme activities were dependent upon dose and time. The effects of the compound were not reversed by concomitant treatment of FG cells with R-5020 or dexamethasone, thus suggesting that RU 486 acted directly on steroidogenic enzymes to alter their activity.

Journal of Endocrinology (1991) 130, 71–78
Osmoregulatory actions of insulin-like growth factor-I in rainbow trout (Oncorhynchus mykiss)
Authors: McCormick S. D. , Sakamoto T. , Hasegawa S. , Hirano T.
Published: December 2008

The ability of insulin-like growth factor-I (IGF-I), insulin and GH to promote hypoosmoregulatory ability was examined in juvenile rainbow trout (Oncorhynchus mykiss). Following adaptation to 12 parts per thousand (p.p.t.) seawater for 5 days, fish were given a single injection of hormone or vehicle, then exposed to 29 p.p.t. for 24 h and examined for changes in plasma osmolarity, ions and glucose. Ovine GH (oGH; 0·2 μg/g) significantly improved the ability of rainbow trout to maintain plasma osmolarity and sodium levels following transfer to 29 p.p.t. seawater. Recombinant bovine IGF-I (0·01, 0·05 and 0·2 μg/g) also improved the hypoosmoregulatory ability of trout; the effect being dose-dependent and greater than that of oGH. Bovine insulin (0·01, 0·05 and 0·2 μg/g) had no statistically significant effect on plasma ions. The results indicate that IGF-I is a potential mediator of the action of GH in seawater adaptation of salmonids.

Journal of Endocrinology (1991) 130, 87–92
Endothelin-like immunoreactivity in rat models of diabetes mellitus
Authors: Takahashi K. , Suda K. , Lam H.-C. , Ghatei M. A. , Bloom S. R.
Published: December 2008

The factors associated with high concentrations of circulating plasma immunoreactive endothelin in patients with diabetes mellitus are unknown. Plasma and tissue (lung and kidney) immunoreactive endothelin levels were therefore measured by radioimmunoassay in three animal models of diabetes mellitus: dexamethasone-treated rats (2 mg/kg per day for 12 days), streptozotocin-treated rats (100 mg/kg, 4 days before being killed) and rats treated with both dexamethasone and streptozotocin. Plasma concentrations of immunoreactive endothelin in the dexamethasone-treated rats (3·13±0·28 pmol/l, mean ± s.e.m., n = 15) were significantly (P < 0·005) higher than those in controls (1·33±0·18 pmol/l, n = 15), while plasma concentrations of immunoreactive endothelin in streptozotocin-treated rats (n = 8) and rats treated with both dexamethasone and streptozotocin (n= 14) were undetectable (< 0·5 pmol/l). Fast protein liquid chromatographic analysis of the plasma immunoreactive endothelin of dexamethasone-treated rats showed four peaks: one in the void volume, one eluting before endothelin-3, one eluting after endothelin-3 and before endothelin-1 and one eluting in a position identical with that of endothelin-1. Pulmonary concentrations of immunoreactive endothelin in the three groups of rats with diabetes mellitus were lower (P < 0·005) but no significant change was found in renal immunoreactive endothelin. These findings indicate that short-term dexamethasone treatment increases plasma levels of immunoreactive endothelin while streptozotocin treatment decreases them. Thus, multiple factors may influence plasma concentrations of immunoreactive endothelin in diabetes mellitus.

Journal of Endocrinology (1991) 130, 123–127
In-vivo inhibition of the preovulatory LH surge by substance P and in-vitro modulation of gonadotrophin-releasing hormone-induced LH release by substance P, oestradiol and progesterone in the female rat
Authors: Battmann T. , Parsadaniantz S. Mélik , Jeanjean B. , Kerdelhué B.
Published: December 2008

The effects of substance P (SP) on the preovulatory surge of LH and on the inhibitory and stimulatory effects of oestradiol-17β and progesterone on gonadotrophin-releasing hormone (GnRH)-induced LH release were investigated in vivo and in vitro in the rat. A single s.c. injection of 100 μg SP at 12.00 h on the day of pro-oestrus significantly decreased the preovulatory surge of LH. In vitro, the inhibitory effect of oestradiol-17β on GnRH-induced LH release was not modified by treatment with SP. The stimulatory effect of progesterone on GnRH-induced LH release was reduced by treatment with SP. It is concluded that SP may play a modulatory role in the neuroendocrine control of the preovulatory LH surge.

Journal of Endocrinology (1991) 130, 169–175
Avidin and ovalbumin induction by progesterone in chicken oviduct detected by sensitive immunoenzymometric assays
Authors: Joensuu T. , Tuohimaa P. , Vilja P.
Published: December 2008

This study describes sensitive immunoenzymometric assays (IEMAs) for chicken avidin and ovalbumin, markers of cytodifferentiation and action of progesterone and oestrogen in the oviduct magnum mucosa. The determination range was 0·5–100 ng/ml and the detection limit 0·1 ng/ml in both IEMAs. The intra- and interassay coefficients of variation, measured from chicken tissue supernatants, averaged below 6 and 10% respectively. IEMAs correlated well with the radioimmunoassays for avidin and ovalbumin previously developed in our laboratory, and with the widely used [14C]biotin-binding method for avidin. Using an IEMA, we found avidin induction with low concentrations of progesterone in the differentiated oviduct of oestrogen-pretreated chicks. The induction has not been detected previously by less sensitive methods. Avidin was induced by all given doses of progesterone (0·2–200 mg/kg in vivo for 24 h after a short oestrogen treatment), the response being dose-dependent at doses of 0·2–20 mg progesterone/kg body weight, the maximum avidin production being about 70 μg/g tissue. Ovalbumin was induced at doses of 2–200 mg progesterone/kg body weight without variations in the responses, being about 35 mg/g. The mean content of avidin in the oviduct of laying hens was 58·1 μg/g, and of ovalbumin 74·9 mg/g. Minimal traces of avidin and ovalbumin were found in the oviduct after hatching (0·3 and 5 μg/g respectively); however, progesterone did not have an effect on this expression. Sensitivity, rapidity and practicability, together with non-radioactivity, are the main advantages of the present IEMAs for chicken avidin and ovalbumin.

Journal of Endocrinology (1991) 130, 191–197
Separation and characterization of Leydig cells and macrophages from rat testes
Authors: Dirami G. , Poulter L. W. , Cooke B. A.
Published: December 2008

A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for 125I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g).

The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH.

It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody.

Journal of Endocrinology (1991) 130, 357–365
The influence of reproductive status on vasopressin release in the rat
Authors: Forsling M. L. , Kelestimur H. , Windle R.
Published: December 2008

It has been shown that surgical ovariectomy of the rat results in a fall in plasma vasopressin concentrations suggesting that ovarian steroids may influence hormone release. To determine whether a similar fall is found on suppression of the oestrous cycle, vasopressin concentrations were monitored after treatment with the antioestrogen preparation tamoxifen or a long-acting analogue of LH-releasing hormone (LHRH) which suppresses ovarian function. Treatment with either agent was found to result in a fall in circulating vasopressin concentrations, with little effect on fluid balance. To determine whether the ovary could influence the vasopressin release in response to known stimuli, hormone concentrations were measured in ovariectomized animals during extracellular fluid hypertonicity produced by an i.p. injection of hypertonic saline and hypovolaemia produced by an i.p. injection of polyethylene glycol. It was found that after ovariectomy or treatment with tamoxifen, the response to hypertonicity was unaffected but that to hypovolaemia was attenuated. Treatment with LHRH affected the response to both hypovolaemia and hypertonicity.

Journal of Endocrinology (1991) 130, 387–393
Administration of insulin-like growth factor-I, but not growth hormone, increases maternal weight gain in late pregnancy without affecting fetal or placental growth
Authors: Gargosky S. E. , Owens J. A. , Walton P. E. , Owens P. C. , Wallace J. C. , Ballard F. J.
Published: December 2008

During late pregnancy in the rat, circulating levels of insulin-like growth factor-I (IGF-I) and some IGF-binding proteins (IGFBP) decline. The aim of the present study was to determine the relationship of GH to circulating IGF and IGFBP in the late-pregnant rat and to examine the effects on maternal, fetal and placental growth of preventing the decline in serum IGF and IGFBP concentrations. During the first 9 days of pregnancy, IGF-I concentrations increased from 340 to 500 μg/l. Recombinant human (rh) GH at 2·4 mg/kg per day and rhIGF-I at 1·4 mg/kg per day were infused into pregnant rats via osmotic mini pumps during the second half of pregnancy. After pump implantation on day 11 of pregnancy, only IGF-I infusion significantly increased circulating IGF-I. A maximum IGF-I concentration of 907 μg/l was measured on day 14 during treatment with IGF-I, after which the serum concentration decreased to 510 μg/l by day 20 of pregnancy. The serum IGFBPs were examined using a Western ligand blot technique. Infusion of neither GH nor IGF-I returned the IGFBPs to non-pregnant levels. Administration of IGF-I slightly increased IGFBP-3 and a smaller 32 kDa IGFBP at days 17 and 20 of pregnancy.

Neither fetal nor placental weight was significantly different between treatment groups. However, administration of IGF-I significantly increased maternal weight gain during the 10-day treatment period. Thus, pregnant rats infused with IGF-I gained 99±4 g (mean ± s.e.m., n = 10) compared with rats treated with GH or vehicle which gained 72±4 g (n = 9) and 77±4 g (n = 10) respectively. The increase in maternal weight after administration of IGF-I was not due to increased litter size, fetal or placental weight. The increased maternal weight gain after IGF administration, without affecting fetal and placental weights, suggests a modification in the mode of maternal nutrient repartitioning during late pregnancy.

Journal of Endocrinology (1991) 130, 395–400
Pharmacological characterization of a novel oestrogen antagonist, ZK 119010, in rats and mice
Authors: Nishino Y. , Schneider M. R. , Michna H. , von Angerer E.
Published: December 2008

The oestrogenic and antioestrogenic effects of a new non-steroidal compound ZK 119010(2-(4-hydroxyphenyl)-3-methyl-1-[6-(1-pyrrolidinyl)-hexyl]-indol-5-ol) were evaluated and compared with those of tamoxifen and ICI 164384.

In immature mice, ZK 119010 administered once daily for 3 days (s.c.) inhibited the uterotrophic and vaginotrophic effect of oestradiol in a dose-dependent manner and was distinctly more potent than tamoxifen or ICI 164384 in exerting antioestrogenic effects.

When antioestrogens in combination with oestradiol were administered once daily for 5 days to ovariectomized adult rats, ZK 119010 at lower doses (≤ 1 mg/kg) was slightly less effective than tamoxifen in inhibiting the uterotrophic effect of oestradiol. At the higher doses, however, ZK 119010 was strongly antioestrogenic, and ICI 164384 was less effective than ZK 119010 or tamoxifen. ZK 119010 at 10 mg/kg, like ICI 164384 at 30 mg/kg, caused an almost complete inhibition of the oestradiol-induced uterine growth in rats. The antioestrogenic effect of tamoxifen in rats was also limited by its inherent oestrogenic property. The oestrogenic activity of ZK 119010 was much below that of tamoxifen, whereas ICI 164384 did not show oestrogenicity. The present results indicate that ZK 119010 is a novel type of non-steroidal antioestrogen which has only a marginal oestrogenic effect in rats and mice. Such an antioestrogen may be useful for the treatment of oestrogen-sensitive diseases in man.

Journal of Endocrinology (1991) 130, 409–414
Long-term effects of recombinant human growth hormone treatment on skeletal maturation and growth in female rhesus monkeys with normal pituitary function
Authors: Wilson M. E. , Tanner J. M.
Published: December 2008

Female rhesus monkeys (n = 5), having normal pituitary function, were treated for 50 months with recombinant human growth hormone (rhGH; 250 μg/kg) 3 days/week (Monday, Wednesday and Friday) and rates of growth were compared with a group of age-matched untreated females (n = 6). Treatment was initiated at 20 months of age, approximately 10 months before the expected age of menarche. Long-term treatment with rhGH accelerated bone maturation and increased the velocity of increase in crown–rump length, tibia length and body weight. The period of acceleration occurred coincident with the occurrence of spontaneous puberty. Body measurements remained larger in the treated females until growth ceased. Long-term rhGH treatment increased final adult crown–rump length by some 3%, with a slight increase in tibia length and body weight, without having any untoward effects on reproductive capacity or health. One treated animal exhibited higher estimates of antibodies to rhGH throughout the study period, and this female also had a smaller increment in crown–rump length than the other treated females. These data suggest that long-term treatment of normal-pituitary females with rhGH augments crown–rump growth without any untoward effects of health.

Journal of Endocrinology (1991) 130, 435–441
Protein glycation: its role in the changes induced by diabetes in the properties of the serum insulin-like growth factor-I binding proteins
Authors: Cortizo A. M. , Gagliardino J. J.
Published: December 2008

The purpose of this work was to study the effect of diabetes on 125I-labelled insulin-like growth factor (IGF) binding to specific serum binding proteins (IGFBPs) and the possible role of protein glycation in such an effect. Accordingly, ligand blotting and fructosamine assays were performed in serum samples from diabetic and non-diabetic eSS rats as well as in samples of normal rat serum previously incubated with different concentrations of glucose.

IGFBPs with molecular weights of 24, 30 and 40 kDa were identified in samples from diabetic and non-diabetic rats. 125I-Labelled IGF-I binding to each of these fractions increased significantly in the serum of diabetic rats. IGF-I binding to IGFBP-40 increased significantly as a function of the degree of glycation of serum proteins. Conversely, the increased binding of IGFBP-24 and IGFBP-30 was related only to the glucose concentration attained at 120 min during the oral glucose tolerance test.

Glycation of proteins of normal serum and the binding of labelled IGF-I increased as a function of glucose concentration in the incubation media. In these in-vitro glycated normal sera, only the binding to IGFBP-40 increased significantly; this increase was closely related to the amount of protein glycation. No clear and reproducible changes occurred with the binding of 125I-labelled IGF-I to IGFBP-24 and IGFBP-30 fractions.

These results confirm the increase in the binding capacity of IGFBPs reported in diabetic animals. They also show that the increase in IGF-I binding to each IGFBP fraction is regulated by a different mechanism; whereas protein glycation induces changes in IGFBP-40, this mechanism does not affect the binding properties of the other two IGFBPs.

The increased binding of IGFBP might affect the availability of free IGF-I, and the consequent alterations in IGF-I-dependent metabolic processes could explain the role of this growth factor in the pathogenesis of chronic complications of diabetes.

Journal of Endocrinology (1991) 131, 33–38
Uptake of nicotinamide by rat pancreatic β cells with regard to streptozotocin action
Authors: Sofue M. , Yoshimura Y. , Nishida M. , Kawada J.
Published: December 2008

Exposure of rat pancreatic β cells in monolayer culture to 2 mmol streptozotocin (STZ)/1 for 1 h followed by thorough washing inhibited their uptake of [14C]nicotinamide and [3H]2-deoxyglucose ([2H]2-DG) to about 50% and also reduced the intracellular ATP concentration to 50% of that in control cells. These changes were not due to a lethal cytotoxic effect of STZ, because cell viability, as estimated by succinic dehydrogenase activity, was 90% of that of control cells. Oligomycin and carbonylcyanide-m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, caused a dose-dependent decrease in intracellular ATP concentration while maintaining high cell viability. These ATP-depleted cells showed a decrease in insulin release and an inhibition of the uptake of [14C]nicotinamide and [3H]2-DG in a dose-dependent manner. Therefore oligomycin and CCCP reproduced the same effects as those found in β cells treated with STZ. These results suggest that the uptake of nicotinamide and 2-DG by β cells might be regulated by their intracellular ATP concentration. The decreased uptake of nicotinamide in ATP-depleted β cells caused by STZ might explain the lack of protective effect of nicotinamide against STZ cytotoxicity when administered after the latter. Furthermore, the radiotracer experiments demonstrated that the transport of nicotinamide by intact β cells was inhibited in a dose-dependent manner by 2-DG and vice versa, i.e. the transport of 2-DG was inhibited by nicotinamide. These findings suggest the existence of a common transport mechanism in β cells responsible for the uptake of nicotinamide and 2-DG, the transport of which is known to occur by facilitated diffusion.

Journal of Endocrinology (1991) 131, 135–138
Parathyroiditis in the non-obese diabetic mouse – a new finding
Authors: Krug J. , Williams A. J. K. , Beales P. E. , Doniach I. , Gale E. A. M. , Pozzilli P.
Published: December 2008

Autoimmune reactions to parathyroid cells have been observed in human autoimmune polyendocrinopathy, but such findings have not been described in animal models of polyendocrine autoimmunity. We report here three cases of lymphocytic infiltrations in 12 parathyroid glands identified in a total of 18 thyroid glands studied in the non-obese diabetic (NOD) mouse.

The majority of parathyroid-infiltrating lymphocytes possessed the helper/inducer phenotype as defined by the L3T4 monoclonal antibody. Parathyroiditis was accompanied by lymphocytic thyroiditis only on one occasion, whereas in other cases of thyroiditis, lymphocytic infiltration of the parathyroid was undetectable. We conclude that parathyroiditis in the NOD mouse is part of the wide spectrum of autoimmunity observed in this animal model of diabetes.

Journal of Endocrinology (1991) 131, 193–196
Oestradiol-17β induces the major vitelline envelope proteins in both sexes in teleosts
Authors: Hyllner S. J. , Oppen-Berntsen D. O. , Helvik J. V. , Walther B. T. , Haux C.
Published: December 2008

During growth of the ovarian follicle, the teleost oocyte becomes surrounded by an acellular coat, the vitelline envelope. The nature, origin and number of the vitelline envelope proteins in fish appear to vary with species. In this work, polyclonal antibodies directed against vitelline envelope proteins from rainbow trout, brown trout and turbot were used to show that oestradiol-17β induces the major vitelline envelope proteins in juveniles, both males and females, from different species. The fact that males can synthesize vitelline envelope constituents shows that the origin of these proteins is not confined to the ovary. The vitelline envelope of rainbow trout eggs consists of three major proteins, designated α (60 kDa), β (55 kDa) and γ (50 kDa). The amino acid composition of each of the three proteins indicated that the three proteins are alike and the suggestion that these proteins represent a separate class of structural proteins is sustained.

Journal of Endocrinology (1991) 131, 229–236
Effect of cell density on growth hormone release from, cyclic AMP levels in and peptide α-amidation activity of primary cultured rat anterior pituitary cells
Authors: Sugimoto H. , Suzuki M. , Takeuchi T. , Ishikawa K.
Published: December 2008

To investigate the effect of cell density on rat somatotrophs, dispersed adult rat anterior pituitary cells were plated at several densities (0·6, 1·2, 2·4 and 6·0 × 105 cells/cm2) or at two densities (6·07 × 105 and 1·21 × 105 cells/cm2) as high and low densities respectively. A static incubation system was used to study the release of GH and peptidyl glycine α-amidating enzyme (PAM; a component of secretory granules) and the cellular concentration of cyclic AMP (cAMP) in basal and stimulated cells. In addition, a perifusion system was used to characterize the sensitivity to GH-releasing factor (GRF) at both high and low densities. The high density of cells in both the static incubation and perifusion systems caused a low basal secretion rate of GH and PAM. When the cultured cells were stimulated with human GRF (hGRF) in perifusion, GH secretion from cells at high density was markedly higher than that from cells at low density. The cAMP content in the static incubation system showed a similar tendency to that observed for basal and stimulated GH secretion; that is, the basal cAMP content was increased as the cell density decreased. The cellular concentration of cAMP was increased by about threefold by 1 nmol hGFR/1 and by more than tenfold by 10 nmol hGRF/1. When the medium from cells cultured at low density was replaced by that from the cells at high density, there was no change in the basal cellular cAMP content of the cells at low density. These data suggest that cell-to-cell contact in dispersed pituitary cells seems to be important in the maintenance of their cellular integrity to secrete GH.

Journal of Endocrinology (1991) 131, 237–244
Rapid effects of parathyroid hormone(1–34) and prostaglandin E2 on bone blood flow and strontium clearance in the rat in vivo
Authors: Cochrane E. , McCarthy I. D.
Published: December 2008

The vascular effects of noradrenaline, ATP, parathyroid hormone (PTH) and prostaglandin E2 (PGE2) were investigated in the rat. Additionally, the exchange of mineral ions between bone and blood was assessed by measuring strontium clearance, with the aim of investigating whether the vascular effects of these agents altered uptake of mineral ions or if this exchange could be changed independently of blood flow. Radioactive microspheres and 85Sr were used to establish bone blood flow and mineral clearance. Measurements of bone blood flow and arterial pressure were made in each animal and used to calculate vascular resistance. A measurement of 85Sr clearance was also obtained. Arterial blood pressure was significantly affected by noradrenaline (P ≤ 0·003) and ATP (P ≤ 0·015). Additionally, noradrenaline significantly (P ≤ 0·03) reduced bone blood flow. This decrease was related to a significant increase in vascular resistance. Arterial blood pressure and bone blood flow were significantly reduced by both bovine PTH(1–34) (P ≤ 0·001, P ≤ 0·02) and PGE2 (P ≤ 0·005, P ≤ 0·001). Vascular resistance to bone was increased by both agents but this was only statistically significant in the case of PGE2 (P ≤ 0·01). A significant (P ≤ 0·001) reduction in strontium was also produced by PGE2. In each group the relationship between bone blood flow and strontium clearance was then analysed. Only the PGE2-treated group had a slope of the regression which was statistically different from both the control animals and the other drug-treated groups. Treatment with PGE2 therefore resulted in a dose-related decrease in 85Sr clearance which was not related to the reduction in bone blood flow.

Journal of Endocrinology (1991) 131, 359–365
Calcium transport in the proximal convoluted tubule and loop of Henle of rats made diabetic with streptozotocin
Authors: Garland H. O. , Harris P. J. , Morgan T. O.
Published: December 2008

In-vivo microperfusion was used to localize the reabsorptive defect responsible for the hypercalciuria of diabetes mellitus and to investigate possible causative factors. Unidirectional proximal calcium absorption was not significantly different in rats made diabetic with streptozotocin compared with controls, providing evidence against the involvement of this nephron segment in the phenomenon. Calcium absorption by the loop of Henle, was however, significantly (P<0·01) lower in diabetic animals (32·1 ±1·2 vs 40·4±0·6 pmol/min). Based on our knowledge of calcium movements within the loop, it is likely that the reabsorptive defect resides within the thick ascending limb. The calcium lesion was found to be independent of acute changes in intraluminal glucose concentration and could not be corrected by acute insulin treatment. The study also provides new information on the relationship between intratubular glucose and fluid movements in the rat nephron. In diabetic rats a proximal perfusate containing 30 mmol glucose/l resulted in fluid absorption comparable with that seen in control rats perfused with 5 mmol glucose/l. However, intraluminal glucose had a stimulatory effect on fluid absorption in the loop of Henle of diabetic rats (10·7 ±0·5 vs 7·9±0·4 nl/min; P<0·01).

Journal of Endocrinology (1991) 131, 373–380