bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the effects of crude and highly purified bovine inhibin (Mr 32 000) on plasma concentrations of FSH and LH in chronically ovariectomized prepubertal heifers
Authors: Beard A. J. , Castillo R. J. , McLeod B. J. , Glencross R. G. , Knight P. G.
Published: December 2008

Chronically ovariectomized prepubertal heifers were used for a comparison of the effects of highly purified bovine inhibin (Mr 32 000) and steroid-free bovine follicular fluid (bFF) on the secretion of FSH and LH. In view of the limited availability of highly purified inhibin, an initial study was undertaken to establish the optimal method for administration of bFF inhibin activity. In comparison with the FSH response to a single large i.v. bolus injection of bFF (50 ml; 3250 mg protein), a far more effective suppression of plasma FSH concentrations was achieved when considerably less bFF (6·3 ml; 410 mg protein) was administered gradually over an extended time-period (2 days) either as a continuous i.v. infusion or as a series of 2-hourly i.v. injections. Following a single i.v. bolus injection of bFF, immunoreactive inhibin was cleared rapidly from the circulation (half-life 51 ± 8 (s.e.m.) min, n = 5), presumably accounting for its limited ability to suppress FSH secretion when administered in this manner.

In a second experiment, treatment of ovariectomized heifers (three per group) with highly purified Mr 32 000 bovine inhibin at a dose rate of 15 μg/2 h for 2 days significantly (P < 0·05) suppressed plasma FSH concentrations, which reached their minimum values (40% suppression) during day 2 of treatment. At a lower dose rate (5 μg/2 h), inhibin did not significantly affect plasma FSH levels. Administration of bFF was also associated with a dose-dependent suppression of FSH secretion. For each of three dose rates tested (three heifers per group), plasma FSH concentrations were maximally suppressed during day 2 of treatment (65 mg/2 h, 86% suppression, P < 0·001; 21·7 mg/2 h, 66% suppression, P < 0·001; 7·2 mg/2 h, 15% suppression, P > 0·05). Neither highly purified inhibin nor bFF significantly affected mean plasma LH concentrations, LH pulse frequency or LH pulse amplitude. Thus we have shown for the first time that highly purified Mr 32 000 bovine inhibin does possess in-vivo biological activity in cattle, promoting a selective suppression of plasma FSH concentrations qualitatively similar to that evoked by steroid-free bFF. Quantitatively, the inhibin preparation had an in-vivo biopotency about 1000 times greater than that of bFF, a value which accords well with its biopotency (1176 × bFF) in the in-vitro rat pituitary cell bioassay.

Journal of Endocrinology (1990) 125, 21–30
Enhancement of bovine growth hormone activity by antibodies against growth hormone peptides
Authors: Bomford R. , Aston R.
Published: December 2008

The biological activity of growth hormones can be enhanced by complexing with monoclonal antibodies of appropriate specificity. In order to define the regions associated with the phenomenon, site-directed antisera to ovine GH (oGH) were prepared by vaccination of sheep with synthetic peptides. Peptides from six distinct regions of the oGH molecule raised antibodies which recognized the hormone in solid-phase radioimmunoassay; however, only one peptide elicited high-affinity antibody as determined by liquid-phase assay. This peptide, corresponding to amino acid sequence 35–53, resulted in circulating hormone antibody in the majority of vaccinated sheep. Immunoglobulin prepared from the serum of immunized animals produced an enhancement of the somatotrophic activity of exogenously administered GH in dwarf mice as determined by the incorporation of [35S]sulphate into costal cartilage. The identification of an antigenic peptide sequence from oGH/bovine GH which elicits enhancing antisera, raises the possibility of a growth-promotion vaccine.

Journal of Endocrinology (1990) 125, 31–38
Effect of cysteamine on the lysosomal enzymes of the hyperprolactinaemic rat pituitary
Authors: Jeitner T. M. , Oliver J. R.
Published: December 2008

The effect of cysteamine on the activity of lysosomal enzymes and the prolactin content of isolated hyperprolactinaemic cells has been investigated. In broken cell preparations, cysteamine markedly stimulated acid prolactin protease activity. In intact cells, however, cysteamine inhibited acid prolactin protease activity and β-galactosidase. Moreover, the activities of α-mannosidase, acid phosphatase, β-glucuronidase, total arylsulphatase and hexosaminidase were not changed by the addition of cysteamine. Cysteamine significantly depleted the cells of prolactin, and this action was not compromized by the inclusion of either leupeptin, chloroquine or NH4Cl in the incubation media. Taken together, these results indicate that cysteamine does not promote degradation of prolactin and hence depletion of prolactin from the pituitary through a mechanism involving lysosomal enzyme degradation.

Journal of Endocrinology (1990) 125, 75–80
Effect of LH injections on testicular steroidogenesis, cholesterol side-chain cleavage P450 mRNA content and Leydig cell morphology in hypogonadal mice
Authors: Scott I. S. , Charlton H. M. , Cox B. S. , Grocock C. A. , Sheffield J. W. , O'Shaughnessy P. J.
Published: December 2008

Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH) and the gonads consequently lack exposure to gonadotrophins during development. We injected male hpg mice with LH for 10 days to investigate whether LH alone can stimulate normal steroidogenesis in these animals. Control animals had an inactive interstitium and very few germ cells. Testicular content of androgens was undetectable by radioimmunoassay in control animals unless a single injection of LH was given 1 h before death, when androgens were just detectable. Control testes incubated in vitro with [3H]pregnenolone demonstrated that without gonadotrophin stimulation pregnenolone was metabolized only to progesterone in significant amounts. Assay for cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA showed basal expression in saline-treated hpg mouse testis. LH treatment induced hypertrophy and hyperplasia of Leydig cells and division of germ cells. Testicular androgen content increased significantly, with testosterone and androstenedione as the major androgens. LH-treated testes incubated with [3H]pregnenolone in vitro had a greater synthetic capacity for testosterone, suggesting an increase in 17α-hydroxylase/C17–20-lyase activity. Basal and human chorionic gonadotrophinstimulated androgen production in vitro increased markedly following LH treatment to levels previously described in the normal adult animal. LH treatment caused a rapid and transient increase in the hybridization of P450scc mRNA which was sevenfold greater than that of saline-treated controls when the animals were killed 1 h after the last injection but fell to control levels within 24 h of cessation of treatment.

We conclude that LH alone can stimulate steroidogenesis in testes of hpg mice, and that previous exposure of the tissue to physiological concentrations of endogenous gonadotrophins is not required to obtain normal steroidogenic responsiveness.

Journal of Endocrinology (1990) 125, 131–138
Circulating levels of GH-releasing hormone and GH during human pregnancy
Authors: Mazlan M. , Spence-Jones C. , Chard T. , Landon J. , McLean C.
Published: December 2008

To study the potential role of GH-releasing hormone (GHRH) in maintaining circulating levels of GH during pregnancy, 302 maternal plasma samples were collected from non-fasted subjects at various stages of pregnancy and assayed for GHRH using a 'two-site' immunoradiometric assay. The GH and placental lactogen levels were also determined. In addition, maternal plasma samples taken during labour, amniotic fluid and cord blood were also assayed for these hormones.

Maternal plasma GHRH levels were similar to non-pregnant levels throughout gestation despite fluctuations in GH values which were always higher than non-pregnant levels. There was no significant difference between GHRH levels in maternal plasma and cord blood although high GH levels were observed in the latter. These findings suggest that peripheral GHRH levels do not play an important role in maintaining circulating GH levels during pregnancy.

Journal of Endocrinology (1990) 125, 161–167
Insulin-like growth factor receptors in chicken liver membranes: binding properties, specificity, developmental pattern and evidence for a single receptor type
Authors: Duclos M. J. , Goddard C.
Published: December 2008

Two distinct receptors for the insulin-like growth factors (IGF-I and IGF-II) have been identified in mammalian tissues, but so far only a receptor structurally related to the type I receptor has been identified in chicken embryonic tissues. This study was designed to characterize binding sites for IGF peptides in chicken liver microsomal membranes prepared from hatch to 10 weeks of age which is the period of most rapid growth.

Binding of both human (h) IGF-I and hIGF-II was displaceable by either peptide and exhibited similar pH, time and temperature dependency. Human IGF-II was more potent than hIGF-I in competing for the binding of the iodinated ligands with half-maximum effective concentrations of 3–5 μg/l and 7–13 μg/l respectively. Porcine insulin was also a potent competitor. Affinity cross-linking studies, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions demonstrated that both IGF peptides were linked to a protein with a molecular weight of about 130 000 Da characteristic of the α-subunit of the type I receptor. There was no evidence for the presence of a type II receptor similar to that found in mammals.

Specific binding of both peptides was low on the day of hatch, increased about threefold by day 3 of age and remained high for the first 3 weeks of life before returning to a lower steady state level up to 10 weeks of age. This was the result of variation in receptor number, with no change in the affinity of the binding site for either ligand. The affinity constant for IGF-II (4·5 ± 0·5 (s.e.m.) litres/nmol) was higher than for IGF-I (1·4 ± 0·3 litres/nmol).

Insulin-like growth factors have previously been reported to stimulate the metabolism and growth of hepatocytes in vitro and are produced by these cells. The occurrence of a higher number of receptors at the period of most rapid growth of the liver suggests that they may have a role in regulating normal liver growth in an autocrine or paracrine manner. Furthermore, present evidence suggests that this is through a single receptor related to the type I receptor.

Journal of Endocrinology (1990) 125, 199–206
Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone
Authors: Houston B. , O'Neill I. E. , Mitchell M. A. , Goddard C.
Published: December 2008

The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chicken GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6·86 and the major peak (cGH P2) an isomer with an isoelectric point of 7·52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (Mr = 23 500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence.

This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l. The output of insulin-like growth factor-I by hepatocytes in culture was increased from a basal rate of 50·4±11·6 (mean ± s.e.m.) to 787·9 ± 98·6 pg/6 × 106 cells per 48 h by two separate pulses of 1 μg cGH P2/ml. An i.v. injection of cGH P2 (15 μg/kg body weight) decreased the thyroxine:tri-iodothyronine ratio in serum of adult hens from 15·71 to 4·44, indicating an increase in 5′-monodeiodinase activity. These results demonstrate that the single most abundant charge isomer of chicken pituitary GH is likely to contain all the biological activity ascribed to the hormone.

Journal of Endocrinology (1990) 125, 207–215
Stimulatory effect of retinoic acid on GH gene expression: the interaction of retinoic acid and tri-iodothyronine in rat pituitary cells
Authors: Morita S. , Matsuo K. , Tsuruta M. , Leng S. , Yamashita S. , Izumi M. , Nagataki S.
Published: December 2008

We have previously demonstrated that retinoic acid (RA) as well as thyroid hormone stimulates GH gene expression. To clarify the relationship between the action of RA and thyroid hormone, pituitary-specific gene expression was investigated further in rat pituitary cells.

Rat clonal pituitary cells, GH3, were treated with RA with or without tri-iodothyronine (T3) for up to 3 days. After treatment with 10–1000 nmol RA/1 with or without 0·1–10 nmol T3/1, medium was collected for radioimmunoassay and cells were subjected to RNA extraction, and GH and prolactin gene expression was analysed using 32P-labelled rat GH and rat prolactin cDNA probes respectively. The data demonstrated the dose–responsive manner of the stimulatory effects of RA and T3 on GH secretion with T3-depleted media. The action of RA was additive to that of T3 for GH secretion when maximum effective doses of RA or T3 were used. Using dot blot and Northern gel analysis, it was shown that RA increased GH mRNA levels in T3-depleted media, and that this action of RA was additive to that of T3 on the induction of GH mRNA levels. In contrast, neither RA nor T3 stimulated the secretion of prolactin and prolactin mRNA levels in these cells.

Our results indicate that RA stimulates GH mRNA increment and GH secretion in T3-depleted media, and that the stimulatory effect of RA is additive to the maximum effective dose of T3.

Journal of Endocrinology (1990) 125, 251–256
The presence of classical insulin-like growth factor (IGF) type-I and -II receptors on mouse osteoblasts: autocrine/paracrine growth effect of IGFs?
Authors: Slootweg M. C. , Hoogerbrugge C. M. , de Poorter T. L. , Duursma S. A. , van Buul-Offers S. C.
Published: December 2008

Specific binding to and proliferative actions of insulinlike growth factors-I and -II (IGF-I and -II) on fetal mouse osteoblasts were tested. Membranes of mouse osteoblasts were shown by binding competition studies to possess specific binding sites for IGF-I and IGF-II. When IGF-I was used as a tracer, half-maximal displacement was obtained with 1·11 μg IGF-I/1 and with 14 μg IGF-II/1. Displacement of 125I-labelled IGF-I was accomplished with 2·33 μg IGF-II/1 and with 55 μg IGF-I/1. Affinity cross-linking showed bands of 130 kDa 125I-labelled IGF-I and 260 kDa 125I-labelled IGF-II under reducing conditions, further indicating the presence of classical type-I and -II receptor sites on mouse osteoblasts. Mitogenic effects of IGFs were weak; a combination with epidermal growth factor or fibroblast growth factor showed strong synergistic action however. The possibility of autocrine/paracrine actions of IGFs is discussed.

Journal of Endocrinology (1990) 125, 271-277
Involvement of the adrenal gland in the suckling-induced decrease in LH and FSH secretion and the concomitant increase in prolactin secretion in the rat
Authors: Taya K. , Sasamoto S.
Published: December 2008

The role of the adrenal gland in the regulation of gonadotrophin and prolactin secretion in the lactating rat was investigated. Changes in secretion of LH, FSH, prolactin, ACTH, β-lipotrophin (β-LPH), inhibin, corticosterone and progesterone after adrenalectomy were examined during the second half of lactation. Follicular maturation was determined by the ability of the follicles to ovulate in response to 10IU human chorionic gonadotrophin (hCG). Adrenalectomy on day 10 of lactation prevented an increase in plasma concentrations of LH and FSH in response to ovariectomy performed at the same time as adrenalectomy, and markedly stimulated secretion of ACTH, β-LPH and prolactin. Adrenalectomy reduced the number of follicles capable of ovulating in response to hCG. Concentrations of inhibin and progesterone in the plasma significantly decreased after adrenalectomy, indicating that development of ovulatory follicles and luteal function had been suppressed. Abolishing the increase in plasma concentrations of LH and inducing a decrease in FSH in the plasma by adrenalectomy therefore prevented maturation of a new set of follicles usually seen during the second half of lactation in rats. The decrease in plasma concentrations of LH also inhibited the ability of the corpus luteum to secrete progesterone, although high concentrations of plasma prolactin were maintained in adrenalectomized lactating rats.

These results indicate that the pituitary-adrenal system is capable of influencing the maintenance of a normal secretion of gonadotrophin and prolactin as well as the maintenance of ovarian function during lactation in the rat.

Journal of Endocrinology (1990) 125, 279—285
Monoclonal antibodies against the free subunits of human chorionic gonadotrophin
Authors: Berger P. , Klieber R. , Panmoung W. , Madersbacher S. , Wolf H. , Wick G.
Published: December 2008

Discordant results on body fluid levels of human chorionic gonadotrophin (hCG) free α- and β-subunits under physiological and pathophysiological conditions, prompted us to raise a total of 260 monoclonal antibodies (MCA) against free hCG-α, free hCG-β, holo-hCG, human follicle-stimulating hormone and bovine luteinizing hormone; 153 MCA recognizing the human α-subunit and 28 reacting with hCG-β were extensively analysed for their intra- and interspecies cross-reactivity with homologous hormones, and for the compatibility of epitopes recognized by them. The immunological topography of free hCG-α and free hCG-β was resolved by these MCA, and epitope maps were designed. Six antigenic determinants on the free α-chain (α1–α6), clustered in three spatially distinct domains, and seven epitopes on the surface of free hCG-β (β1–β7), could be distinguished. Strikingly, three α-chain epitopes (α4, α5 and α6) were shared between various species, which is in contradiction to the concept of immunological species-specificity of α-subunits. Three determinants were found to be present only on the free subunits but not on holo-hCG (α6, β6 and β7), and only two determinants (β1 and β7) were hormone-specific for hCG. Based on this information, an immunoenzymometric assay for the free α-subunit of human glycoprotein hormones was established, with a sensitivity of 1·3 pg/well and a cross-reactivity with holo-hCG of less than 0·005% Thus this assay provides the basis for detecting free α-subunits in the presence of extremely high levels of holo-hormones, which may assist in elucidating the role of free α-subunits in man.

Journal of Endocrinology (1990) 125, 301–309
Dose—response curves for treatment with biosynthetic human growth hormone
Authors: Darendeliler F. , Hindmarsh P. C. , Brook C. G. D.
Published: December 2008

We have studied the effect of varying doses of biosynthetic human GH on the growth response over the first year of therapy in 90 short prepubertal children (67 males and 23 females, aged 3·1–12·9 years). As pretreatment height velocity was the predominant determinant of response, the children were divided into three groups on the basis of their pretreatment height velocity standard deviation scores (SDS). Group A (n=27) had pretreatment height velocity SDS between +1·3 and −0·8 (short normal growing children), group B (n=35) between −0·9 and −2·0 (moderate GH insufficiency) and group C (n=28) less than −2 (severe GH insufficiency). Within each group, the dose of GH administered was the dominant factor in the regression. At a mean dose of 16 units/m2 body surface area per week, the change in height velocity SDS over the first year of therapy was +2·9 (95% confidence interval (CI) 2·4, 3·5) in group A, +4·3 (95% CI 3·9, 4·6) in group B and +7·0 (95% CI 6·0, 8·0) in group C. These values translate into increases in growth rate of 2·2, 3·4 and 5·5 cm/year for an 8-year-old in each group respectively. These results have important implications in planning the treatment of children with GH insufficiency.

Journal of Endocrinology (1990) 125, 311–316
Secretion of ACTH by perifused isolated rat anterior pituitary cells: pulses of secretagogue enhance the secretory response and modify the effect of atriopeptin
Authors: Antoni F. A. , Dayanithi G.
Published: December 2008

The aim of the present study was to characterize the inhibitory action of atriopeptin on secretagogue-evoked ACTH release in vitro.

Perifused isolated rat anterior pituitary cells were exposed to repeated pulses of 41-residue corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP). The net ACTH secretory response to both neurohormones increased progressively with the number of pulses applied, until a maximum hormonal response was reached which was stable for the subsequent period of observation (2–3 h). The maximal secretagogue-evoked hormone release eventually achieved was 4 and 1·7 times greater than the initial response to AVP and CRF-41 respectively. The size of the ACTH response elicited by 50 pmol CRF-41/1 and 500 pmol AVP/1 (CRF/AVP) given together also underwent progressive enhancement. The number of secretagogue pulses required to reach the maximal response to a particular stimulus depended upon the concentration of the secretagogue peptides, higher concentrations favoured a more rapid development of the stable secretory response.

The potency of 103–126 residue atriopeptin to inhibit CRF/AVP-induced ACTH release varied by about 1000-fold depending upon the prior treatment of the cells. In general, cells not previously exposed to secretagogues appeared largely resistant, those under a moderate secretagogue drive were strongly inhibited, and those under intense stimulation were again refractory to inhibition by atriopeptin. In contrast, corticosterone suppressed stimulated ACTH release regardless of the state of the cells.

The data demonstrate that the conditions of cell maintenance are pivotal determinants of the inhibitory effect of atriopeptin on secretagogue-stimulated ACTH release in vitro. The in-vivo correlate of these findings may be that the sensitivity of corticotrophs towards atriopeptin is determined by requirements encoded through the pattern of release of hypothalamic CRF-41 and AVP.

Journal of Endocrinology (1990) 125, 365–373
Effects of gastric inhibitory polypeptide, vasoactive intestinal polypeptide and peptide histidine isoleucine on the secretion of hormones by isolated mouse pancreatic islets
Authors: Bailey C. J. , Wilkes L. C. , Conlon J. M. , Armstrong P. H. , Buchanan K. D.
Published: December 2008

The release of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) by isolated mouse pancreatic islets was determined during 30-min incubations at 5.6 and 16.7 mmol glucose/l in the absence and presence of gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) at concentrations of 1–1000 nmol/l. Insulin release was enhanced (>50%) by GIP (100–1000 nmol/l) and VIP (1 μmol/l) at 5.6 mmol glucose/l, but not at 16.7 mmol glucose/l. Glucagon release was increased by GIP (100–1000 nmol/l), and by VIP and PHI (1—1000 nmol/l) at both glucose concentrations in a dose-related manner (maximum increases > tenfold). Somatostatin release was similarly increased by GIP (10–1000 nmol/l) at both glucose concentrations. Only the highest concentration (1 μmol/l of PHI tested increased somatostatin release (twofold) at 5.6 mmol glucose/l, whereas PHI and VIP (1–1000 nmol/l reduced (>37%) somatostatin release at 16.7 mmol glucose/l. PP release was increased (49–58%) by 100–1000 nmol GIP/l, but was not significantly altered by VIP, and was reduced (39–56%) by PHI. The results indicate that GIP, VIP and PHI each stimulate glucagon release in a dose-related manner, but they exert discretely different effects on other islet hormones depending upon the dose and the prevailing glucose concentration.

Journal of Endocrinology (1990) 125, 375–379
Effects of passively immunizing ewes against inhibin and oestradiol during the follicular phase of the oestrous cycle
Authors: Mann G. E. , Campbell B. K. , McNeilly A. S. , Baird D. T.
Published: December 2008

Passive immunization was used to investigate the importance of inhibin and oestradiol in the control of FSH production during the follicular phase of the oestrous cycle in the sheep. Four groups of five mature Scottish Blackface ewes were injected with normal sheep plasma (control), antiserum to the 1–26α peptide fragment of porcine inhibin, antiserum to oestradiol-17β, or a combination of the two antisera, 24 h following cloprostenol-induced luteal regression. There was no difference in the concentration of LH in jugular venous plasma between the control and inhibin-immunized groups following the injection of normal sheep plasma or inhibin antiserum, with both groups exhibiting normal LH surges. In both the groups immunized against oestradiol, the basal concentration of LH rose by 25–30% (P<0.05) during the 96-h period following injection, while the LH surge and consequent formation of a corpus luteum was inhibited. In all three immunized groups there was a significant (P<0.001) rise in the concentration of FSH starting 3.8–4.8 h after the injection of antiserum. The duration of the rise was similar in the groups injected with oestradiol antiserum alone (43.6±12.8 h) or in combination with inhibin antiserum (40.6 ± 11.7 h), but was significantly (P<0.05) shorter in the group immunized against inhibin alone (17.0 ± 0.5 h). The rise in FSH was similar in the groups immunized against inhibin (142 ± 6%) or oestradiol (143±4%) alone, and was significantly (P<0.01) greater in the group injected with both antisera (195± 17%). These results provide evidence that both oestradiol and inhibin play a role in regulating the concentration of FSH during the follicular phase of the oestrous cycle, while reinforcing the hypothesis that inhibin is not involved in the regulation of LH production.

Journal of Endocrinology (1990) 125, 417–424
Serotonin enhances oestradiol production by hamster preovulatory follicles in vitro: effects of experimentally induced atresia
Authors: Terranova P. F. , Uilenbroek J. Th. J. , Saville L. , Horst D. , Nakamura Y.
Published: December 2008

Preovulatory follicles from adult hamsters on the morning of pro-oestrus were used in this study. Serotonin stimulated oestradiol production by preovulatory follicles during a 5-h incubation in 1 ml Krebs–Ringer bicarbonate glucose medium containing isobutylmethylxanthine (0.1 mmol/l; IBMX) and androstenedione (1 μmol/l). The enhanced oestradiol production by serotonin was dependent on the dose of IBMX and androstenedione. Mianserin, a serotonin type-1 and serotonin type-2 receptor antagonists, prevented the serotonin-enhanced oestradiol production in a dose-dependent manner. Ketanserin, a specific serotonin type-2 receptor antagonist, was ineffective in blocking the action of serotonin, indicating that the effect of serotonin was mediated by the serotonin type-1 receptor. In the presence of androstenedione (1 μmol/l), serotonin was unable to enhance oestradiol production in isolated granulosa cells. It was also unable to enhance oestradiol production in early atretic follicles; atresia was induced experimentally by an injection of phenobarbital in order to prevent ovulation.

The data indicate that serotonin stimulates oestradiol production by hamster preovulatory follicles in vitro. The mechanism of action of serotonin involves an intact healthy follicle, a serotonin type-1 receptor and possibly cyclic AMP. The increased oestradiol secretion might be related to increased androgen production by the follicle and increased permeability (leakiness) of the follicle to androstenedione which serves as substrate for aromatization to oestradiol by the granulosa cell.

Journal of Endocrinology (1990) 125, 433–438
Human chorionic gonadotrophin-like and β-core-like materials in postmenopausal urine
Authors: Akar A. H. , Gervasi G. , Blacker C. , Wehmann R. E. , Blithe D. L. , Nisula B. C.
Published: December 2008

In addition to human chorionic gonadotrophin (hCG), the urine of pregnant women contains a small molecular weight form of the hCG-β subunit known as β-core. Human CG-like material has been described in tissues, serum and urine of normal man, particularly in postmenopausal women. We examined different urine preparations from postmenopausal women to determine whether β-core-like material, as well as hCG-like material, could be detected. We studied an acetone extract of a pool of 11 litres of postmenopausal urine, three different commercial preparations of human menopausal gonadotrophins and two commercial preparations of 'pure' FSH. After Sephadex G-100 chromatography of these various postmenopausal urine extracts, fractions were assayed using four assay systems to detect hCG, β-core, LH and FSH immunoreactivities. Human CG immunoreactivity was readily detected in all urinary extracts and it eluted in a position indistinguishable from that of purified hCG. In addition to this hCG-like material, all urinary extracts, except the commercial 'pure' FSH preparations, contained material which reacted in the β-core radioimmunoassay. This β-core immunoreactive material eluted from Sephadex G-100 in a position corresponding to that of purified pregnancy-derived β-core. We conclude that the urine of postmenopausal women contains material resembling the β-core molecule found in pregnancy urine. The origin of this β-core-like material remains to be determined, and its presence will have an impact on the application of urinary β-core as a tumour marker.

Journal of Endocrinology (1990) 125, 477–484
Localization and characterization of epidermal growth factor receptors on human testicular tissue by biochemical and immunohistochemical techniques
Authors: Stubbs S. C. , Hargreave T. B. , Habib F. K.
Published: December 2008

In the present study attempts were made to characterize the epidermal growth factor (EGF) receptor on human testicular tissue. A radioligand exchange assay with 125I-labelled EGF was used to detect a high affinity, low capacity, single binding site in the 105 000 g particulate fraction of human testicular tissue. Binding was optimal at 32 °C following a 40-min incubation with a mean ( ± s.d.) dissociation constant of 327 ±59 pmol/l (d.f. 9). The number of binding sites ranged from 0.07 to 0.21 pmol/mg protein. Competition studies with other peptide hormones including LH, FSH, prolactin, insulin-like growth factor-I, fibroblast growth factor and nerve growth factor have confirmed the specificity of EGF for its receptor. The receptor was also found to be heatlabile and sensitive to trypsinization. Cross-linking experiments using disuccinimidyl suberate revealed major binding species at the 125 kDa region and this is thought to represent a proteolysed form of the receptor. Immunohistochemical localization of the receptors demonstrated their presence in the interstitial tissue and not within the seminiferous tubules. The presence of specific EGF binding in the interstitial tissue suggests that EGF may play some role in testicular steroidogenesis.

Journal of Endocrinology (1990) 125, 485–492
Patterns of variation in progestin in pregnant sheep
Authors: Caton D. , Kalra P. S.
Published: December 2008

Patterns of variation in progestin concentrations were studied in pregnant sheep during the last half of gestation. In three sets of experiments, blood was drawn daily or every 4 h for 24 h or every 10 min for 8 h. Peripheral arterial concentration of progestin was examined for 'peaks' using the Pulsar program which identified peaks in daily samples and in samples collected every 10 min, though less often than in daily samples. Ovariectomy did not affect the frequency or character of the peaks. The data suggest that large variations in progestin concentrations occur during the last half of pregnancy. They are superimposed on a gradual increase in average concentration and last for 2 to 3 days.

Journal of Endocrinology (1990) 126, 37–42
Increased in-vivo insulin sensitivity but normal liver insulin receptor kinase activity in dwarf chickens
Authors: Guéritault I. , Simon J. , Chevalier B. , Derouet M. , Tixier-Boichard M. , Mérat P.
Published: December 2008

The effects of the recessive and sex-linked dw gene on insulin sensitivity and liver insulin receptors were compared in normal (Dw-dw) and dwarf (dw-dw) brother or half-brother chickens. At 3·5 weeks of age, following an overnight fast, exogenous insulin (0–6·9 nmol/kg body weight) was slightly but significantly more hypoglycaemic in dwarf chickens. At 4 weeks of age, following an oral glucose load (2 g/kg), glucose tolerance was the same in both genotypes, whereas plasma insulin levels were greatly decreased in dwarf chickens. At 5 weeks of age, plasma concentrations of glucose and insulin were the same in both genotypes in the fasting state and decreased in the fed state in dwarf chickens. In liver membranes prepared from fasted chickens, insulin binding was increased in dwarf chickens, while the affinity of insulin receptors and the insulin-degrading activity of the membranes were the same in both genotypes. Following solubilization with Triton X-100, liver receptors were successively purified on lentil then wheat germ lectins. Autophosphorylation of the β-subunit did not differ between either the genotype or the nutritional (fed or fasted) state. In the basal state (in the absence of insulin) the tyrosine kinase activity of the receptor towards artificial substrate poly(Glu,Tyr)4:1 was significantly decreased in dwarf chickens by fasting. However, the change in tyrosine kinase activity of the receptor in response to insulin was similar, irrespective of the genotype and the nutritional state. Therefore, the slight increase in insulin sensitivity observed in vivo in dwarf chickens is accounted for, at least partly, by a slight increase in liver insulin receptor number, but not by a change in the kinase activity of liver insulin receptors. In addition, post-insulin receptor kinase events and/or GH-dependent counter-regulatory mechanisms may superimpose and increase the insulin sensitivity of dwarf chickens.

Journal of Endocrinology (1990) 126, 67–74
Potential mechanism of insulin resistance in ageing: impaired insulin-stimulated glucose transport due to a depletion of the intracellular pool of glucose transporters in Fischer rat adipocytes
Authors: Matthaei S. , Benecke H. , Klein H. H. , Hamann A. , Kreymann G. , Greten H.
Published: December 2008

To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (30MG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 30MG (12±4 vs 13±3 fmol/5 × 104 cells, means ± s.e.m.); in contrast, in FR-24 rats insulin-stimulated 30MG was markedly decreased by 43% when compared with that in FR-6 rats (158±14 vs 90±8 fmol/5 × 104 cells; P < 0·01). Insulin binding to adipocytes from FR-6 rats was 2·40±0·38% compared with 2·28±0·47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48·6±7·2 vs 29·8±5·5 pmol/mg membrane protein; P < 0·01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23·9±4·2 (FR-6) vs 14·4±3·1 (FR-24) pmol/mg membrane protein; P < 0·01). These results were confirmed by immunoblotting.

In conclusion, (1) maximal insulin-stimulated 30MG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 30MG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 30MG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats.

Journal of Endocrinology (1990) 126, 99–107
Purification and structural characterization of ovine placental lactogen
Authors: Warren W. C. , Liang R. , Krivi G. G. , Siegel N. R. , Anthony R. V.
Published: December 2008

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4·2 mg of oPL, with an ∼ 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0·18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4·1 nmol/l) and oPRL (ED50 = 1·1 μmol/l) in competing for 125I-labelled oPL-binding sites.

Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a λZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides). The predicted amino acid sequence derived from the nucleotide sequence confirmed homology to bPL (67%) and oPRL (48%). Little amino acid sequence existed with other PLs (≤29%) or GH proteins (≤27%). These results suggest that oPL and oGH are more biologically similar in their ability to compete for fetal liver binding sites, but that oPL is structurally more similar to oPRL. Elucidation of exact structure–function relationships for oPL will, however, require further investigation.

Journal of Endocrinology (1990) 126, 141–149
Development of adenylate cyclase activity in rat adrenal glands during the perinatal period
Authors: Chatelain A. , Naaman E. , Durand P. , Lepretre A. , Dupouy J. P.
Published: December 2008

Adenylate cyclase activity was studied in crude adrenal membranes from fetal and newborn rats. Basal adenylate cyclase activity was higher in fetal than in newborn rats. ACTH(1–24) (1 μmol/l), guanosine (β,γ-imido diphosphate) (Gpp(NH)p) (10 μmol/l) and forskolin (100 μmol/l) stimulated the activity of the enzyme at all stages studied. The sensitivity of the enzyme to ACTH was maximal on days 17 and 19 of gestation. When Gpp(NH)p was added to ACTH(1–24), the response was significantly higher than that induced by Gpp(NH)p alone. Forskolin and Gpp (NH)p alone increased the adenylate cyclase activity and the sensitivity of the enzyme to these compounds was higher in newborn rats than in fetuses. Treatment of 21-day-old rat fetuses with ACTH increased the response of adenylate cyclase to Gpp(NH)p alone or to forskolin whereas treatment with dexamethasone did not modify the response of the enzyme to either Gpp(NH)p alone or forskolin. Our results show that the change in the responsiveness of adenylate cyclase takes place immediately after birth during the first week and ACTH is able to induce a maturation of the fetal adrenal adenylate cyclase system.

Journal of Endocrinology (1990) 126, 211–216
Short-loop inhibition of thyrotrophin-releasing hormone-induced growth hormone secretion in fowl
Authors: Lea R. W. , Ahene C. A. , Marsh J. A. , Harvey S.
Published: December 2008

The i.c.v. administration of 0·1 or 10 μg ovine (o)GH to 12- to 16-week-old hypothyroid chickens of a sex-linked dwarf (SLD) strain suppressed the basal plasma GH concentrations, measured 24 h afterwards. The GH response of the oGH-injected SLDs to TRH was suppressed, in a dose-related way, in comparison with that induced by TRH in birds given control injections (10 μg) of bovine serum albumin (BSA). Basal circulating concentrations of GH in euthyroid K strain birds of the same age were even lower than in the SLDs following injection of 10 μg oGH, and were not further reduced by oGH administration. The GH response to TRH in the K strain birds injected i.c.v. with 0·1 or 10 μg oGH was, nevertheless, suppressed in comparison with the BSA-injected K strain controls. The i.c.v. administration of oGH also suppressed circulating concentrations of LH and the LH response to TRH in the K strain birds.

Twenty-four hours after i.c.v. administration of oGH (10 μg), the somatostatin (SRIF) content in the medial basal hypothalamus of 8-week-old euthyroid cockerels was greater than that in BSA (10 μg)-injected controls. At the same time, the binding of [3H]3-methyl-histidine2-TRH to the pituitary caudal and cephalic lobes of GH-injected birds was less than that in the controls.

These results suggest that GH regulation in avian species is partly mediated by an inhibitory short-loop mechanism (mediated by hypothalamic SRIF and a down-regulation of pituitary TRH-binding sites) that suppresses basal and secretagogue-induced GH release.

Journal of Endocrinology (1990) 126, 237–244
Thyrotrophin increases the α1b-adrenergic receptors in rat thyroid gland in vivo
Authors: Shimura H. , Endo T. , Onaya T.
Published: December 2008

Using chlorethylclonidine (CEC), an αlb-adrenergic receptor-selective antagonist, we characterized α1-adrenoceptor subtypes in rat thyroid gland, and investigated the effect of methimazole (MMI)-induced high TSH levels on α1 receptor subtypes and noradrenaline-induced iodide organification. The density of thyroid α1-adrenergic receptors was increased about sixfold in rats treated with MMI for 3 weeks compared with controls. Pretreatment of thyroid membrane preparations with CEC (10 μmol/l) caused an 83% decrease in specific 2-[β-(hydroxy-3-[125I]iodophenyl) ethylaminomethyl]tetralone binding sites in MMI-treated rats, but only a 43% decrease in control rats. The density of CEC-insensitive α1 receptors (α1a) was similar in MMI-treated and control rats, so MMI was shown to increase CEC-sensitive α1 receptors (α1b).

Noradrenaline-stimulated iodide organification was threefold greater in MMI-treated rats than in control rats when values were expressed as a per cent increase over basal levels. Pretreatment of thyroid lobes with 10 μmol CEC/1 for 30 min caused a 66% decrease in maximal noradrenaline-induced iodide organification in MMI-treated rats, but a significantly lower decrease (49%) in control rats.

These results suggest that the rat thyroid gland contains both α1a and α1b receptors, both of which mediate noradrenaline-induced iodide organification, and also that TSH enhances noradrenaline-induced iodide organification by increasing α1b receptor density.

Journal of Endocrinology (1990) 126, 317–322
Influence of oestrogen and progesterone on macrophage distribution in the mouse uterus
Authors: De M. , Wood G. W.
Published: December 2008

Macrophages are constituents of all normal connective tissue including the murine uterus. Macrophages have been identified previously in endometrium and myometrium of pregnant and non-pregnant murine uterus using antibodies against macrophages. In the current study immunohistochemical analysis of murine uterus demonstrated that there were not significant quantitative differences in uterine macrophages between the dioestrous, pro-oestrous and oestrous stages. However, distributional changes occurred during the oestrous cycle. Macrophages were evenly distributed throughout uterine tissue during dioestrus, while, during pro-oestrus and oestrus, their concentration was highest in the subepithelial stroma. Because the oestrous cycle is hormonally regulated, we asked whether or not oestrogen and/or progesterone might influence macrophage distribution. Ovariectomy, which eliminates cyclical production of oestrogen and progesterone, resulted in a significant decrease in both the relative and the absolute number of uterine macrophages within 6 days. Injections of progesterone or oestrogen to ovariectomized mice resulted in restoration of uterine macrophage numbers. Injection of oestrogen plus progesterone in a regimen known to prepare the uterus for receptivity for blastocyst implantation increased the number of macrophages to levels which were consistently higher than those seen during oestrus. Moreover, following hormone administration macrophages were more concentrated in the subepithelial stroma, a distributional pattern which was most evident following injection of both hormones. The results suggest that both oestrogen and progesterone promote quantitative and distributional changes in the uterine macrophage population.

Journal of Endocrinology (1990) 126, 417–424