bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of islet hormones on insulin secretion from cloned B cell lines, HIT-T15 and RINm5F
Authors: Lambert D. G. , Atkins T. W.
Published: December 2008

The effects of the islet cell hormones glucagon, somatostatin-28 and pancreatic polypeptide on insulin secretion from cultured cloned pancreatic B cells (HIT-T15 and RINm5F) have been investigated. Glucagon stimulates the secretion of insulin from HIT-T15 cells in the absence and presence of glucose and from RINm5F cells in the absence and presence of glyceraldehyde. HIT-T15 cells were more sensitive to the stimulatory effect of glucagon than RINm5F cells. Somatostatin-28 and pancreatic polypeptide, both alone and in combination, reduced glucose- and glucagon-stimulated insulin release from HIT-T15 cells and glyceraldehyde- and glucagon-stimulated insulin release from RINm5F cells. HIT-T15 cells were more sensitive to the inhibitory actions of somatostatin-28 and pancreatic polypeptide than RINm5F cells. This study supports the hypothesis that insulin release from normal B cells may be modified by the paracrine activity of islet hormones, glucagon, somatostatin and pancreatic polypeptide and probably occurs before any fine tuning imposed by subsequently released insulin.

Journal of Endocrinology (1989) 121, 479–485
Peripheral blood levels of immunoreactive inhibin during pseudopregnancy, pregnancy and lactation in the rat
Authors: Taya K. , Komura H. , Watanabe G. , Sasamoto S.
Published: December 2008

Concentrations of inhibin in peripheral blood were determined by radioimmunoassay in pseudopregnant, pregnant and lactating rats and correlated with the status of follicular development, luteal function in the ovaries and placental function. In pseudopregnant rats, plasma concentrations of inhibin remained relatively high from days 2 to 12 of pseudopregnancy and no significant changes occurred throughout pseudopregnancy. In pregnant rats, a marked decline in plasma concentrations of inhibin occurred between days 6 and 14 followed by an abrupt increase on days 20 and 21. In lactating rats, plasma concentrations of inhibin were consistently lower in dams nursing eight pups than in dams nursing two pups. Plasma concentrations of inhibin remained low during the early stages of lactation followed by a significant increase during the late stages in dams nursing eight pups. These changes in inhibin during the period of lactation correlated with those of plasma concentrations of LH, but not with those of oestradiol-17β, testosterone and progesterone. Throughout pseudopregnancy, pregnancy and lactation, on the other hand, plasma concentrations of FSH were within the range of dioestrous levels.

These findings indicate that concentrations of inhibin in peripheral plasma vary with the number of healthy antral follicles and no positive correlation was observed with luteal function during the luteal phase in rats. The results also suggest that inhibin is a more direct index of follicular development than oestradiol-17β in rats.

Journal of Endocrinology (1989) 121, 545–552
Nocturnal changes in serum melatonin during female puberty in rhesus monkeys: a longitudinal study
Authors: Wilson M. E. , Gordon T. P.
Published: December 2008

Diurnal concentrations of serum melatonin were determined longitudinally in female rhesus monkeys throughout sexual maturation to ascertain how levels varied with advancing age and reproductive onset. Females were housed either in outdoor enclosures (n = 8) exposed to ambient environmental conditions, or indoors (n = 4) under a photoperiod of 12 h light: 12 h darkness and fixed temperature of 20–23 °C. Animals were studied from immaturity (15 months) through first ovulation and were additionally compared with fully adult female rhesus monkeys (n = 5) studied during the annual breeding season. The diurnal melatonin pattern was described for the developing females in the summer, autumn and winter in 3 successive years from samples collected at 10.00, 18.00, 22.00, 02.00, 06.00 and 10.00 h.

Nocturnal levels of melatonin declined significantly during development in both indoor- and outdoor-housed females with a progressive decrease up to 33 months of age. Daytime values were consistently low but exhibited a slight decline also with age. Nocturnal values in all months sampled fell significantly with greater decreases occurring at the earliest ages. Furthermore, superimposed upon this developmental change, animals housed outdoors responded to seasonal changes in photoperiod with diurnal increases in melatonin occurring after sunset. The females in the present study exhibited first ovulation at two distinct ages: 32–37 months ('early', n = 6) and 41–45 months ('later', n=5). One female did not ovulate within the study period. Although nocturnal levels of serum melatonin were similar between the two groups up to 29 months of age, a post-hoc analysis revealed that concentrations were significantly lower by 34 months of age for the early group, a time coincident with first ovulation. Nocturnal levels of melatonin remained high, relative to the early group, in the later ovulating females until 43 months of age, coincident with first ovulation for these animals. The diurnal pattern of serum melatonin at first ovulation, regardless of chronological age, was similar to that observed during the ovulatory season for adult female rhesus monkeys.

These data suggest that nocturnal melatonin concentrations decline with advancing chronological age in prepubertal female rhesus monkeys. Furthermore, the timing of sexual maturation was inversely related to nocturnal melatonin. Whether this change in melatonin is causally related to reproductive onset or, rather, is a consequence of other factors regulating the occurrence of first ovulation remains to be determined. Furthermore, the observation that the melatonin rhythm in outdoor-housed females follows the prevailing photoperiod permits the hypothesis that this rhythm may mediate any photoperiodic effect on the seasonal occurrence of first ovulation characteristic of rhesus monkeys housed outdoors.

Journal of Endocrinology (1989) 121, 553–562
Thyrotrophin (TSH)-binding proteins in bacteria and their cross-reaction with autoantibodies against the human TSH receptor
Authors: Byfield P. G. H. , Davies S. C. , Copping S. , Barclay F. E. , Borriello S. P.
Published: December 2008

A screen of a range of bacteria normally found in gut flora identified eight with the ability to bind TSH specifically. These included the previously reported Yersinia enterocolitica, Gram-positive, Gramnegative, pathogenic and commensal organisms. Eleven preparations of TSH-receptor autoantibodies strongly able to displace 125I-labelled TSH from the mammalian TSH receptor differed in their ability to displace the tracer from binding to bacterial extracts. None could displace the tracer from E. coli 06–1, four displaced 125I-labelled TSH from E. coli V21/1 and five displaced the tracer from Y. enterocolitica. Of those immunoglobulin preparations which did react with the bacterial protein, their apparent potency compared with that of TSH in displacing tracer from bacterial binders was an order of magnitude greater than with the mammalian receptor. This is consistent with the autoantibodies having a relatively better fit with the bacterial antigen than with the receptor when compared with TSH. The bacterial-binding activity and mammalian receptor-binding activities in each of two samples co-chromatographed on a Remazol yellow GGL–Sepharose affinity column strongly indicated that the same immunoglobulin species reacts with both antigens. These results are consistent with the proposal that a bacterial protein is the primary immunogen for the TSH-receptor antibodies in at least some patients with Graves' disease.

Journal of Endocrinology (1989) 121, 571–577
Controls of corticotrophin-releasing factor output by hypothalamic tissue from fetal sheep in vitro
Authors: Brooks A. N. , Power L. A. , Jones S. A. , Yang K. P. , Challis J. R. G.
Published: December 2008

Corticotrophin-releasing factor (CRF) is thought to be an important physiological regulator of the pituitary-adrenal axis in fetal sheep and, as such, plays a fundamental role in the initiation of parturition in this species. However, little is known of the controls of CRF secretion from the fetal hypothalamus.

We looked for the presence of CRF in fetal hypothalami, and examined whether the hypothalamic CRF concentration or molecular species changed in relation to gestational age. We established an in-vitro perifusion system to examine the release of CRF from perifused hypothalami taken from fetuses at day 100 and day 140 of pregnancy, under basal conditions and in response to potassium depolarization and/or dexamethasone administration.

Immunoreactive CRF was present in fetal hypothalami as early as day 100 (2·42 ± 0·99 (s.e.m.) μg/g protein, n = 9) and in similar concentrations at day 140 (2·31 ± 0·69 μg/g protein, n = 9). There was a significant (P < 0·05) increase in hypothalamic CRF content to 14·79 ± 4·09 μg/g protein (n = 16) between day 122 and day 135 of gestation. Using Sephadex G-75 chromatography, hypothalamic extracts at day 100, days 122–135 and day 140 eluted with a single peak of immunoreactivity which corresponded to synthetic ovine CRF(1–41).

The basal release of CRF from perifused hypothalami at day 140 (76·6 ± 10·4 pg/fraction, n = 8) was significantly (P < 0·05) greater than at day 100 (50·1 ± 10·2 pg/fraction, n = 11). Dexamethasone significantly inhibited basal CRF release at day 140 of gestation but not at day 100. Potassium depolarization caused a rapid release of CRF in all cases, a response which was independent of gestational age or treatment with dexamethasone.

We conclude that the fetal hypothalamus contains immunoreactive CRF as early as day 100 of gestation and that this material may be released when perifused in vitro under basal conditions and in response to a depolarizing agent. The basal release of CRF from perifused hypothalami of day-140 fetuses was greater than at day 100 and was inhibited by dexamethasone, suggesting maturation of negative feedback control of CRF output between days 100 and 140. Since dexamethasone had no effect on potassium-stimulated release of CRF, we suggest that its effects are at sites other than the hypothalamic CRF nerve terminals.

Journal of Endocrinology (1989) 122, 15–22
Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells
Authors: Selden R. F , Yun J. S. , Moore D. D. , Rowe M. E. , Malia M. A. , Wagner T. E. , Goodman H. M.
Published: December 2008

A mouse metallothionein-I/human growth hormone fusion gene was microinjected into fertilized mouse eggs, the embryos were implanted into pseudopregnant foster mothers, and the offspring analysed. Five of twenty-six mice born after one series of injections contained from one to eight copies of the fusion gene stably integrated into their genomes and had human growth hormone in their serum. When several of these transgenic mice and transgenic offspring were treated with glucocorticoids, serum growth hormone levels were elevated from 1·5- to 6·3-fold. A fourfold induction in fusion gene mRNA in the liver of one of the five mice was also observed after treatment with glucocorticoids. When the fusion gene was transiently transfected into mouse L cells, dexamethasone caused a three- to fourfold induction of fusion gene mRNA and secreted human growth hormone. A deletion analysis of regulatory elements required for inducibility in L cells shows that DNA sequences responsible for the observed inductions are located within the transcribed region of the human growth hormone gene. However, a previously described glucocorticoid receptor binding site in the first intron of the gene is not required for response to the hormone.

Journal of Endocrinology (1989) 122, 49–60
The oestrogen-induced surge of LH requires a 'signal' pattern of gonadotrophin-releasing hormone input to the pituitary gland in the ewe
Authors: Clarke I. J. , Cummins J. T. , Jenkin M. , Phillips D. J.
Published: December 2008

Two experiments were conducted with ovariectomized and hypothalamo-pituitary disconnected (HPD) ewes to ascertain the pattern of inputs, to the pituitary gland, of gonadotrophin-releasing hormone (GnRH) necessary for the full expression of an oestrogen-induced LH surge. The standard GnRH replacement to these sheep was to give pulses of 250 ng (i.v.) every 2 h; at the onset of experimentation, pulses were given hourly. In experiment 1, groups of sheep (n = 7) were given an i.m. injection of 50 μg oestradiol benzoate, and after 10 h the GnRH pulse frequency or pulse amplitude was doubled. Monitoring of plasma LH concentrations showed that a doubling of pulse frequency produced a marked increase in baseline values, whereas a doubling of amplitude had little effect on the LH response. In a second experiment, ovariectomized HPD sheep that had received hourly pulses of GnRH for 16 h after an i.m. injection of oil or 50 μg oestradiol benzoate were given either a 'bolus' (2·25 μg GnRH) or a 'volley' (500 ng GnRH pulses 10 min apart for 30 min, plus a 500 ng pulse 15 min later). Both groups then received GnRH pulses (250 ng) every 30 min for the next 13 h. Oestrogen enhanced the LH responses to the GnRH treatments, and the amount of LH released was similar in ovariectomized HPD ewes given oestrogen plus bolus or volley GnRH treatments and ovariectomized hypothalamopituitary intact ewes given oestrogen.

These results suggest that the oestrogen-induced LH surge is initiated by a 'signal' pattern of GnRH secretion from the hypothalamus.

Journal of Endocrinology (1989) 122, 127–134
Neurotransmitters and lymphatic-vascular transfer of prostaglandin F2α stimulate ovarian oxytocin output in sheep
Authors: Heap R. B. , Fleet I. R. , Davis A. J. , Goode J. A. , Hamon M. H. , Walters D. E. , Flint A. P. F.
Published: December 2008

The mechanisms of lymphatic-vascular transfer across the ovarian vascular pedicle were studied in anaesthetized sheep 8–15 days after ovulation. [3H]Prostaglandin F2α (PGF2α), [14C]mannitol and [36Cl]Na were infused continuously into either a uterine lymphatic or a uterine vein and the kinetics of transfer into the adjacent utero-ovarian vein or ovarian plasma were studied. Transfer occurred according to the sequence [36Cl] > [14C] > [3H] indicating that PGF2α is not transferred by rapid diffusion, as with [36Cl]Na, nor by a paracellular route, as with [14C]mannitol, but by a slower process probably involving facilitated diffusion.

Transfer into the adjacent utero-ovarian vein or ovarian blood was greater when compounds were infused into a uterine lymphatic than into a uterine vein. Substantially more [3H]PGF2α occurred in the adjacent corpus luteum than either of the other compounds after a lymphatic infusion. Intra-lymphatic infusion of PGF2α stimulated the release of ovarian oxytocin but the effect was not confined to the adjacent ovary. Intravenous (jugular) infusion of PGF2α failed to stimulate ovarian oxytocin secretion whereas close-arterial infusion into the ovaries was effective, and the possibility was investigated that any systemic effect of PGF2α was mediated through neural mechanisms. Noradrenaline and acetylcholine were both effective in causing the release of ovarian oxytocin when infused close-arterially into the ovary. With infusions of acetylcholine, ovarian oxytocin secretion rate was increased over fivefold without any change in posterior pituitary release. Noradrenaline and acetylcholine produced a concomitant fall in ovarian blood flow, and neurotransmitter-induced ischaemia may have played a role in ovarian oxytocin release. The finding that PGF2α infused into a uterine lymphatic stimulates ovarian secretion of oxytocin, and that the effect is bilateral whereas PGF2α accumulation in ovarian tissue is unilateral, implies that its mechanism of action may not be solely directed at the luteal cell.

Journal of Endocrinology (1989) 122, 147–159
Opioid receptors on cells of the immune system: evidence for δ- and κ-classes
Authors: Carr D. J. J. , DeCosta B. R. , Kim C.-H. , Jacobson A. E. , Guarcello V. , Rice K. C. , Blalock J. E.
Published: December 2008

Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d1. A δ-class opioid receptor was found with an Mr of 58 000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T celland B cell-enriched populations. Using the central (brain) κ-selective agonist, U-69,593, it was also determined that P388d1 cells possess κ-like opioid receptors. Scatchard analysis of the binding of [3H]U-69,593 revealed a single population of sites with a dissociation constant of 17 ± 3 (s.e.m.) nmol/l and a total number of binding sites of 53·8 ± 1·0 (s.e.m.) fmol/106 cells. Moreover, the racemic κ-selective agonist U-50,488H was able to displace 50% of [3H]U-69,593 binding at 8·0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (δ-selective) and [d-Ala2,N - Me - Phe4,Gly5 - ol] - enkephalin (μ - selective) were ineffective displacers of [3H]U-69,593 except at high concentrations.

Journal of Endocrinology (1989) 122, 161–168
Interleukin-1 inhibits thyrotrophin-induced human thyroglobulin gene expression
Authors: Yamashita S. , Kimura H. , Ashizawa K. , Nagayama Y. , Hirayu H. , Izumi M. , Nagataki S.
Published: December 2008

The regulation of thyroglobulin (Tg) and its specific mRNA by interluekin-1 (IL-1) in cultured human thyrocytes was investigated. Specific binding of 125I-labelled IL-1 on thyrocytes was confirmed by solid-phase binding assay. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without recombinant human IL-1. TSH stimulated Tg release from cultured human thyrocytes in a dose- and time-dependent manner. Both IL-1α and β inhibited TSH-induced Tg release at concentrations ranging from 0·01 to 10 U/ml. The suppressive activities of IL-1α and β were similar. They did not alter the basal level of Tg release. Unstimulated human thyrocytes did not contain any detectable Tg mRNA, but TSH-stimulated thyrocytes expressed a single species of Tg mRNA (8·5 kb). Both IL-lα and β inhibited TSH-induced Tg mRNA in a dose-responsive manner. IL-1 (10 U/ml) caused maximal suppression of TSH-induced Tg mRNA to nearly basal levels. In contrast, the γ-actin mRNA hybridization signal was not altered in control or treated cells. Furthermore, IL-1 stimulated [3H]thymidine uptake into thyrocyte DNA. These results demonstrate that IL-1 directly inhibits TSH-induced Tg gene expression and provide further support for a functional role of IL-1 as a local modulator of thyroid hormone synthesis.

Journal of Endocrinology (1989) 122, 177–183
Metformin treatment of lean and obese Zucker rats modulates the ability of glucagon and insulin to regulate hepatocyte adenylate cyclase activity
Authors: Gawler D. J. , Wilson A. , Houslay M. D.
Published: December 2008

Glucagon stimulated adenylate cyclase activity some 21-fold in liver membranes from lean (Fa/Fa) and some 20-fold in membranes from obese (fa/fa) Zucker rats, with constants yielding half-maximal activation (Ka values) of 12·6 and 120·1 nmol/l respectively. Treatment of animals with the biguanide drug metformin (N′,N′-dimethylbiguanide) decreased the ability of glucagon to stimulate this enzyme to some 16-fold for both the lean and obese animals and reduced the Ka values for activation of this enzyme by glucagon to 6·3 and 60·9 nmol/l respectively. Insulin inhibited glucagon-stimulated adenylate cyclase activity by some 24% in liver membranes from lean animals and some 17% in liver membranes from obese animals, with constants yielding half-maximal inhibition (Ki values) of 110 and 160 nmol/l respectively. The ability of insulin to inhibit the adenylate cyclase activity, from obese but not lean animals, was attenuated when insulin concentrations over 5 nmol/l were employed. Treatment of animals with metformin profoundly altered the sensitivity of adenylate cyclase to inhibition by insulin, with inhibition being increased to some 32% using liver membranes from either lean or obese animals. Values of Ki for this inhibitory action of insulin were 520 and 500 nmol/l using membranes from the lean and obese animals respectively, and no reduction in the ability of insulin, at concentrations over 5 nmol/l, to inhibit adenylate cyclase activity was observed using membranes from obese animals. Metformin also changed the kinetics of inhibition of adenylate cyclase by insulin. These were apparently negatively co-operative, with Hill coefficients of 0·76 and 0·89 using membranes from the untreated lean and obese animals respectively, and positively co-operative, with Hill coefficients of 1·45 and 1·20 for the metformin-treated lean and obese animals respectively.

Journal of Endocrinology (1989) 122, 207–212
Production of parathyroid hormone-like peptide in a human osteosarcoma cell line: stimulation by phorbol esters and epidermal growth factor
Authors: Rodan S. B. , Wesolowski G. , Ianacone J. , Thiede M. A. , Rodan G. A.
Published: December 2008

A clonal cell line (Saos-2/B-10) derived from human osteosarcoma Saos-2 cells had the same osteoblastic characteristics as the mother line, but lacked sensitivity to parathyroid hormone (PTH) at early passages. At later passages (> 70) the cells became very sensitive to PTH (0·1 nmol/l). The absence of PTH-stimulatable adenylate cyclase correlated with the secretion of an adenylate cyclase-stimulatory activity which had the properties of the recently characterized PTH-like peptide (PTH-LP). This activity was inhibited by the PTH antagonist [8norleucyl, 18norleucyl, 34tyrosinyl]bovine PTH-(3–34)amide and could be neutralized by an antiserum raised against the synthetic PTH-LP-(1–34). Hybridization with a human PTH-LP cDNA showed that these cells produce two PTH-LP mRNAs of approximately 1·5 and 1·8 kb. The production of PTH-LP was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA; 150 nmol/l) and epidermal growth factor (EGF; 10 ng/ml). The increased accumulation of PTH-LP in conditioned media in response to TPA was seen after 1 h and levelled off at 6 h. In contrast, EGF stimulation was lower at 3 and 6 h but continued for 24 h. Both agents increased PTH-LP mRNA levels in Saos-2/B-10 cells. A TPA analogue which does not stimulate protein kinase C had no effect on PTH-LP production. Cycloheximide blocked the stimulatory effect of both TPA and EGF and the TPA effect was blocked by actinomycin D, suggesting transcriptional control. The regulation of PTH-LP by these agents may offer clues regarding the association of this protein with malignancy.

Journal of Endocrinology (1989) 122, 219–227
Actions of parathyroid hormone-related protein on the rat kidney in vivo
Authors: Zhou H. , Leaver D. D. , Moseley J. M. , Kemp B. , Ebeling P. R. , Martin T. J.
Published: December 2008

Peptides containing residues 1–34 of parathyroid hormone-related protein (PTHrP) and of bovine parathyroid hormone (bPTH), and recombinant full-length PTHrP(1–141) were infused i.v. into anaesthetized thyroparathyroidectomized rats to compare their action and potency on the renal handling of calcium, phosphate and cyclic AMP (cAMP) in vivo. All three peptides decreased the excretion of calcium and increased the excretion of phosphate and cAMP in the urine, with PTHrP(1–34) and PTHrP(1–141) having virtually equipotent effects. Thus the essential requirements for the major physiological activity of PTHrP on the kidney are contained within the 34 amino-terminal amino acids. For all three peptides, the lowest infusion rate that increased phosphate and cAMP excretion was 0·01 nmol/kg per h, whereas the lowest infusion rate that decreased calcium excretion was 0·025 nmol/kg per h for the PTHrP peptides and 0·1 nmol/kg per h for bPTH(1–34). The response to the PTHrP peptides was maximal at an infusion rate of 01 nmol/kg per h for both calcium and phosphate. Since the kidney is either equally sensitive to PTHrP and bPTH(1–34), or more sensitive to PTHrP than to bPTH(1–34), the hypercalcaemia of humoral hypercalcaemia of malignancy may develop because uncontrolled secretion of PTHrP increases the renal reabsorption of calcium to such an extent that even a modest increase in the inflow of calcium into the blood raises plasma calcium concentration.

Journal of Endocrinology (1989) 122, 229–235
Episodic secretion of inhibin into the ovarian vein during the follicular phase of the oestrous cycle in the ewe
Authors: McNeilly A. S. , Baird D. T.
Published: December 2008

Changes in the plasma concentration of inhibin were measured by radioimmunoassay in ovarian venous blood collected at 10-min intervals for 5-h periods between 16 and 21 h and 40 and 45 h after cloprostenol-induced luteal regression in six Finn–Merino sheep. Episodes of inhibin secretion occurred with an interpulse interval of 66 ± 5 min in both stages of the follicular phase. These changes in inhibin were unrelated to pulses of LH or oestradiol. There was no relationship with plasma concentrations of FSH, which did not change in a pulsatile manner. These results suggest that the release of inhibin by the preovulatory follicle(s) occurs in a pulsatile manner and is under local control by unknown factors.

Journal of Endocrinology (1989) 122, 287–292
Inhibin and activin-like activity in fluids from male and female gonads: different molecular weight forms and bioactivity/immunoactivity ratios
Authors: Grootenhuis A. J. , Steenbergen J. , Timmerman M. A. , Dorsman A. N. R. D. , Schaaper W. M. M. , Meloen R. H. , de Jong F. H.
Published: December 2008

The existence of various molecular weight forms of inhibin in ovarian follicular fluid has been reported earlier, while there is no information on the form of inhibin in testicular tissue. Inhibin bioactivity was therefore estimated in eluates of slices, obtained after SDS-PAGE of rat testicular and ovarian homogenates, rat Sertoli cell-conditioned medium (rSCCM) and bovine ovarian follicular fluid (bFF).

The only form of inhibin detected in testes from 22-day-old rats and in rSCCM was a 30 kDa protein. In rat ovarian extracts, larger forms of inhibin were also found as well as the predominant 30 kDa form. An activin-like activity was found in the 25 kDa SDS-PAGE eluates of both rSCCM and ovarian homogenates, which caused a dose-dependent increase of FSH release from cultured pituitary cells.

Activin-like activity and several forms of inhibin were found in bFF after SDS-PAGE. After purification of inhibin from bFF using dye affinity, anion-exchange, lentil lectin affinity chromatography and a subsequent reversed phase chromatography step, two pools of inhibin activity were obtained. These were separated by SDS-PAGE revealing 30 and 58 kDa inhibin forms. The immunoactivity of these forms of inhibin was then estimated using antibodies against the 22 N-terminal amino acids of the α subunit of 30 kDa bovine inhibin. It appeared that the two molecular weight forms of inhibin had bioactivity/immunoactivity ratios which differed more than five-fold. This indicates that results of radioimmunoassays of inhibin of ovarian origin, using peptide antisera, should be interpreted with caution.

Journal of Endocrinology (1989) 122, 293–301
Cellular basis of luteal steroidogenesis in the human ovary
Authors: Fisch B. , Margara R. A. , Winston R. M. L. , Hillier S. G.
Published: December 2008

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20α-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.

Journal of Endocrinology (1989) 122, 303–311
The inter-relationships between ovarian-independent growth, tumorigenicity, invasiveness and antioestrogen resistance in the malignant progression of human breast cancer
Authors: Clarke R. , Brünner N. , Thompson E. W. , Glanz P. , Katz D. , Dickson R. B. , Lippman M. E.
Published: December 2008

Among the processes contributing to the progressive acquisition of the highly malignant phenotype in breast cancer are ovarian-independent growth, antioestrogen resistance and increased metastatic potential. We have previously observed that increased invasiveness and development of ovarian-independent growth occur independently. In an attempt to define the inter-relationships between these processes further, we have compared the phenotypes of ovarian-independent, invasive and antioestrogen-resistant sublines of the ovarian-dependent human breast cancer cell line MCF-7. Cells acquiring ovarian-independent growth can retain sensitivity to anti-oestrogens. One clone of MCF-7 cells selected for stable antioestrogen resistance has become non-tumorigenic but its invasive potential remains unaltered. Thus, acquisition of some characteristics of the progressed phenotype can occur independently. This phenomenon of independent parameters in phenotypic progression could partly explain the considerable intra- and intertumour heterogeneity characteristic of breast tumours.

Journal of Endocrinology (1989) 122, 331–340
Ultrastructural changes induced by oestradiol-17β, progesterone and oestrone-3-sulphate in guinea-pig endometrial glandular cells grown in primary culture
Authors: Alkhalaf M. , Chaminadas G. , Propper A. Y. , Adessi G. L.
Published: December 2008

The effects of oestradiol-17β, progesterone and oestrone-3-sulphate were studied in primary cultures of guinea-pig endometrial glandular epithelial cells. Comparative ultrastructural studies were performed by means of transmission electron microscopy on cells grown either without hormones or with oestradiol-17β (2 nmol/l), oestradiol-17β (2 nmol/l) plus progesterone (50 nmol/l), or oestrone sulphate (0·1 μmol/l). In the control medium, without steroid hormones, the majority of epithelial cells were poorly differentiated, although numerous small mitochondria were present and abundant lipid droplets could be observed. Oestradiol-17β stimulated metabolic activity in the cells. Progesterone added to oestradiol-17β-primed cells stimulated the development of the endoplasmic reticulum–Golgi system. Oestrone sulphate induced a higher level of differentiation characterized by large clear mitochondria, well-developed Golgi complexes, and active nuclei, suggesting secretory activity. In all cases, the cultured cells displayed deep invaginations of the nuclear membrane associated with nuclear pores, known as nucleolar channels. After treatment with oestrone sulphate these channels were associated with a characteristic reticular nucleolus. We conclude that cultured endometrial epithelial cells display secretory activity in response to treatment with oestradiol-17β plus progesterone, or with oestrone sulphate.

Journal of Endocrinology (1989) 122, 439–444
Characterization of oxytocin receptors in rat adenohypophysis using a radioiodinated receptor antagonist peptide
Authors: Chadio S. E. , Antoni F. A.
Published: December 2008

Oxytocin may function as a hypothalamic releasing hormone for prolactin and ACTH secretion in the rat. In the present study we have investigated the properties of putative oxytocin receptors in the rat adenohypophysis by radioligand-binding assay.

A novel oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid),2-(ortho-methyl)-Tyr2-Thr4-Orn8-Tyr9-NH2]-vasotocin (OTA) was radioiodinated by the iodogen method to a specific activity of 0·6 nCi/fmol. The radioiodinated derivative 125I-labelled OTA (125I-OTA) was reacted with membrane suspensions prepared from the uterus or adenohypophysis of female rats which were (a) ovariectomized for 7 days, (b) ovariectomized and treated with 5 μg oestradiol-17β 48 h before death or (c) implanted with a piece of silicone elastomer tubing containing 50 mg diethylstilboestrol (DES) 5 days before death. In uterine as well as the pituitary membrane suspensions, the radioligand was bound reversibly and with high affinity (dissociation constants 0·2 ± 0·1 and 0·1±0·01 nmol/l respectively; means + s.e.m., n=3) to a single class of sites with limited binding capacity, which varied with the type of pretreatment. Oestradiol-17β increased the binding capacity fivefold in the uterus in ovariectomized rats, but only very low specific radioligand binding was found in pituitary preparations from the same animals. Treatment with DES markedly increased the number of receptors in both the uterus and the adenohypophysis. Studies with several agonist and antagonist analogues revealed no difference in the ligand specificity of the uterine and adenohypophysial sites binding 125I-OTA, indicating that they are the same species of receptor. Furthermore, ligand-binding studies, carried out with tritiated vasopressin as tracer in pituitary membrane preparations, showed that OTA is not bound by pituitary vasopressin receptors at concentrations below 1 μmol/l.

In summary, 125I-OTA is a highly specific radioligand suitable for the analysis of pituitary oxytocin receptors. The low number of oxytocin receptors in the adenohypophysis of rats treated with oestradiol-17β suggests that the prolactin-stimulating action of oxytocin is mediated by highly efficient transmembrane signalling.

Journal of Endocrinology (1989) 122, 465–470
Burmese Russell's viper venom causes hormone release from rat pituitary cells in vitro
Authors: Hart G. R. , Proby C. , Dedhia G. , Yeo T. H. , Joplin G. F. , Burrin J. M.
Published: December 2008

Acute and chronic hypopituitarism is associated with severe envenoming by the Burmese Russell's viper. We have demonstrated that in vitro, Burmese Russell's viper venom (0·1–10 μg/ml) causes a dose-dependent release of GH, TSH and ACTH from dispersed rat anterior pituitary cells in culture. At 10 μg/ml, venom causes a significant increase in the release of GH (344%, P<0·001), TSH (168%, P<0·005) and ACTH (>700%, P<0·001). We have also shown that the component (or components) responsible for this stimulatory effect is stable to heat (60 °C, 1 h) and mild trypsinization. Repeated addition of venom (1 μg/ml) to pituitary cells in a perifusion column system demonstrated attenuation of GH release. This reduced response was not due to depletion of the GH pool since the pituitary cells were subsequently able to respond to both GH-releasing factor (GRF) stimulation and KCl depolarization. Somatostatin in a dose which abolished GRF-stimulated GH release failed to affect venom-stimulated GH release, implying that venom acts in a cyclic AMP-independent manner. We conclude that Burmese Russell's viper venom has direct effects on pituitary hormone release in vitro. Whether these effects contribute to its known actions in vivo on the function of the pituitary remains to be established.

Journal of Endocrinology (1989) 122, 489–494
Evidence for differences in low density lipoprotein processing by porcine granulosa and luteal cells in vitro: effect of addition of serum for plating of granulosa cells on lipoprotein metabolism
Authors: Rajkumar K. , Ly H. , Schott P. W. , Njaa B. , Murphy B. D.
Published: December 2008

The present studies were carried out to compare the low density lipoprotein (LDL) metabolism by freshly isolated immature porcine granulosa cells with that by luteal cells. Furthermore, we have examined the effect of serum used for plating of granulosa cells on lipoprotein degradation and utilization. In incubation studies, addition of LDL as an exogenous substrate had a mild stimulatory effect on progesterone accumulation by granulosa cells, while it exhibited a dose-dependent stimulatory effect on luteal cells. When granulosa and luteal cells were incubated with 125I-labelled LDL, membrane binding of LDL occurred in both cell types, but only luteal cells were capable of internalizing the bound LDL. Granulosa cells in incubation degraded LDL much less in comparison with luteal cells, and the amount varied with the maturity of the cells. When granulosa cells were plated with graded amounts of serum which was withdrawn for 48 h following plating, they exhibited enhanced LDL degradation in a serum concentration-dependent fashion. Addition of serum for plating selectively enhanced utilization of LDL, but not high density lipoprotein (HDL) for progesterone accumulation by the cells in culture. Time-course studies on LDL degradation by granulosa cells following serum withdrawal indicate that the ability of cells to degrade LDL decreased in a time-dependent fashion. Serum withdrawal selectively decreased utilization of LDL but not HDL for progesterone secretion. It is concluded that immature granulosa cells have a limited capability to utilize cholesterol carried by LDL. However, when cultured in the presence of serum, cells acquire the ability to utilize more efficiently LDL-carried cholesterol for progesterone secretion which is then lost following long-term withdrawal of serum from culture.

Journal of Endocrinology (1989) 122, 557–564
Post-somatostatin rebound secretion of growth hormone is dependent on growth hormone-releasing factor in unrestrained female rats
Authors: Sugihara H. , Minami S. , Wakabayashi I.
Published: December 2008

To examine the characteristics of GH secretion following the termination of the infusion of somatostatin, unrestrained adult female Wistar rats were subjected to repeated infusions of somatostatin separated by 30-min control periods. When somatostatin was infused for 150 min at a dose of 3, 30 or 300 μg/kg body wt per h, the magnitude of the rebound GH secretion increased in a dose-dependent manner. The infusion of somatostatin at a dose of 300 μg/kg body wt per h for 60, 150 or 240 min progressively augmented the size of the rebound GH secretion. When an antiserum to rat GH-releasing factor (GRF) was injected i.v. 10 min before the end of the infusion, the peak amplitude of the rebound GH secretion (300 μg/kg body wt, 150 min) was reduced to less than 20% of that of control rats. The rebound GH secretion (300 μg/kg body wt per h, 150 min) was augmented by a bolus injection of human GRF (1 μg/kg body wt). The combined effect of the end of infusion of somatostatin and a bolus injection of GRF on the amount of GH secreted was additive. The plasma GH response to GRF was completely inhibited when human GRF (3 μg/kg body wt per h) and somatostatin (300 μg/kg body wt per h) were infused simultaneously for 150 min. The magnitude of the rebound GH secretion following the termination of the co-administration was larger than that following the somatostatin infusion alone, but this rebound was not enhanced by a bolus injection of human GRF. Moreover, the amount of GH secreted was significantly less than that after the termination of somatostatin infusion plus a bolus injection of human GRF in the absence of preceding GRF administration.

These results suggest that at least part of the influence of somatostatin on GH secretion is exerted at the level of the hypothalamus through modulating the release of GRF. In addition, it is inferred that the simultaneous infusion of GRF and somatostatin induces the attenuation of the GH response to GRF through a receptor effect.

Journal of Endocrinology (1989) 122, 583–591
Effect of ovarian hormones, pregnancy and lactation on somatostatin and substance P in the alimentary tract of mice
Authors: Bailey C. J. , Conlon J. M. , Flatt P. R.
Published: December 2008

Immunoreactive somatostatin and substance P were determined in extracts of alimentary tract (stomach to colon) from the following groups of adult female mice: intact control, ovariectomized, ovariectomized and treated with oestradiol (50 μg/kg per day) and/or progesterone (2 mg/kg per day) for 30 days, 19-day-pregnant, and 10-day-postpartum lactating. Ovariectomy increased the somatostatin concentration of the stomach (by 52%, P < 0·05), jejunum (by 116%, P < 0·01) and caecum (by 114%, P < 0·01). These effects were partially or totally prevented by the oestradiol and progesterone treatments, especially the oestradiol-progesterone combination, except for an increase (by 126%, P < 0·01) in gastric somatostatin after treatment with oestradiol alone. Lactation also increased gastric somatostatin (by 108%, P < 0·001), but the somatostatin concentration of other regions of the alimentary tract (jejunum to colon) was reduced (by 21–55%, P < 0·05) in pregnant and lactating mice. The concentration of substance P was increased by ovariectomy in stomach (by 69%, P < 0·01), duodenum (by 84%, P < 0·05), ileum (by 163%, P < 0·001) and caecum (by 57%, P < 0·01). This effect was partially or totally prevented by treatment with progesterone alone and by the oestradiol-progesterone combination, but not by oestradiol alone. Pregnancy and lactation increased gastric substance P by 46% (P < 0·01) and 61% (P < 0·001) respectively, but substance P concentrations in other regions of the alimentary tract were not significantly altered. The results suggest that ovarian oestrogens and progestogens are important in the maintenance of normal concentrations of somatostatin and substance P in the gastrointestinal tract of female mice.

Journal of Endocrinology (1989) 122, 645–650
Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat
Authors: Harbuz M. S. , Lightman S. L.
Published: December 2008

In-situ hybridization histochemistry was used to measure corticotrophin-releasing factor mRNA and proenkephalin A mRNA in the paraventricular nucleus (PVN), and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary of the rat. Levels of message were determined at 1, 2, 4 and 8 h after exposure to a variety of physical and psychological stresses. Corticotrophin-releasing factor mRNA in the PVN and POMC mRNA in the anterior pituitary increased in response to i.p. hypertonic saline, restraint and swim stress but not to cold stress. Proenkephalin A mRNA was raised only in response to the physical stress of i.p. injection of hypertonic saline. These results suggest that different afferent pathways and hypothalamic neurotransmitters may be involved in mediating the hypothalamic response to different physical and psychological stresses.

Journal of Endocrinology (1989) 122, 705–711
Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe
Authors: Ayad V. J. , Wathes D. C.
Published: December 2008

Oxytocin-binding sites in the endometrium and myometrium of the non-pregnant ewe were characterized. [3H]Oxytocin bound to a single site in both tissues with high affinity; dissociation constants were determined to be 1·96 nmol/l in endometrium and 2·12 nmol/l in myometrium. Oxytocin binding was enhanced by divalent cations with a similar order of potency in both tissues: Co2+> Mn2+> Ni2+> Mg2+ > Zn2+> Ca2+. The endometrial and myometrial binding sites showed the same specificity for oxytocin analogues and related peptides, having high affinity for oxytocin, [Arg8]-vasopressin, [Lys8]-vasopressin, and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4,Gly7]-oxytocin. The results suggest that oxytocin receptors present in the endometrium and myometrium of the ewe are similar both to each other and to classical oxytocin receptors.

Journal of Endocrinology (1989) 123, 11–18
Relationship between growth hormone-releasing hormone and somatostatin in the rat: effects of age and sex on content and in-vitro release from hypothalamic explants
Authors: Ge F. , Tsagarakis S. , Rees L. H. , Besser G. M. , Grossman A.
Published: December 2008

Secretion of GH in the rat has been shown to be dependent upon age and sex. Using rat hypothalamic explants in vitro, we have studied the release and hypothalamic content of GH-releasing hormone (GHRH) and somatostatin in male and female Wistar rats at four different ages (10, 30 and 75 days, and 14 months). Basal release of GHRH was not significantly different between male and female rats, but at all ages males released more GHRH in response to stimulation by both 28 and 56 mmol potassium/l than female rats (P<0·05). Neither basal nor potassium-stimulated release of GHRH altered with age. In contrast, both basal and potassium-stimulated secretion of somatostatin increased significantly (P<0·01) with age, but was the same in the two sexes. Hypothalamic GHRH content, as assessed by the extractable tissue content following incubation, was significantly (P<0·01) lower in 10-day-old rats compared with older rats, but remained constant after 30 days of age. Somatostatin content, in contrast, increased progressively with age (P<0·01). The hypothalamic content of the two peptides was the same in both sexes.

In conclusion, our findings demonstrate that male rats release more GHRH in vitro than female rats, possibly reflecting the increased pulse amplitude of GH seen in males in vivo; the progressive fall in secretion of GH previously reported during ageing appears to parallel the progressive increase in somatostatin release and content seen in our in-vitro system.

Journal of Endocrinology (1989) 123, 53–58