bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of the administration of human chorionic gonadotropin to postnatal rats
Authors: Gaytan F , Pinilla L , Romero J L , Aguilar E
Published: December 2008

Neonatal and prepubertal male rats were treated with human chorionic gonadotropin (hCG, 5 IU/g body weight per day) on days 2–4 or 20–22. Depending on the date of treatment, different groups of rats were sacrificed at 5, 23, 30 and 100 days of age, in order to study the short-and long-term effects of the treatment with hCG on the development of the testes and sex accessory organs. Rats treated with hCG on days 2–4 showed increased number and size of foetal Leydig cells at 5 days of age. However, long-term effects include decreased numbers of adult-type Leydig cells, decreased weight of the testes and sex accessory organs, decreased basal and hCG-stimulated testosterone secretion, and delayed balano-preputial separation. In contrast, animals treated with hCG on days 20–22 showed similar short- and long-term effects, consisting of increased number of adult-type Leydig cells and macrophages, increased weight of the testes and sex accessory organs and advanced balano-preputial separation. In adulthood, both groups showed normal reproductive function.

These results seem to indicate that the effects of hCG treatment in prepubertal rats are dependent on the type of Leydig cell stimulated, and suggest that foetal Leydig cells play a regulatory role in the early postnatal testicular development.

Journal of Endocrinology (1994) 142, 527–534
Improvement of mild hypoinsulinaemic diabetes in the rat by low non-toxic doses of vanadate
Authors: Henquin J C , Carton F , Ongemba L N , Becker D J
Published: December 2008

To exert their anti-diabetic effects in animals with overt alterations of glucose homeostasis, vanadium salts must be administered in high doses, which also cause decreases in food intake and body weight gain. In this study, we evaluated the effect of low doses of vanadate in rats made mildly diabetic (fed plasma glucose levels ∼11 mmol/l) and moderately hypoinsulinaemic by the injection of streptozotocin 4 days after birth. Vanadate was added to food and drinking water, at concentrations that led to the consumption of about 1 mg vanadium element per day (∼2·65 mg vanadium/kg per day in adult rats), i.e. three to fivefold less than in previous studies. The treatment was started at weaning and lasted 22 weeks (V rats), or was administered for 9 weeks only from the age of 3 months (C-V rats). Food intake and body weight gain were not affected in V rats and decreased by no more than 10% in C-V rats. In V rats, fasted and fed plasma glucose levels were decreased by about 0·5 and 2–3 mmol/l, respectively. The rises in glycaemia after three oral glucose tolerance tests were also clearly attenuated. These effects were not accompanied by any changes in plasma insulin levels. Pancreatic insulin reserves (decreased by two-thirds as compared with normal rats) were not affected by the treatment. A decrease in plasma glucose levels was also noted in C-V rats, and this improvement disappeared upon cessation of the treatment. In conclusion, oral vanadate improves glucose homeostasis in rats with moderate insulin deficiency and diabetes, even when the element is administered at low doses which have practically no repercussions on food intake and body weight gain. This indicates that vanadium salts have genuine beneficial effects on glucose metabolism in vivo.

Journal of Endocrinology (1994) 142, 555–561
Insulin-like growth factors-I and -II and their binding proteins during postnatal development of dwarf Snell mice before and during growth hormone and thyroxine therapy
Authors: van Buul-Offers S C , Bloemen R J , Reijnen-Gresnigt M G , van Leiden H A , Hoogerbrugge C M , Van den Brande J L
Published: December 2008

The ontogeny of serum insulin-like growth factors (IGFs)-I and -II and their binding proteins (IGFBPs) was studied in normal and dwarf Snell mice. IGF-I concentrations in serum of normal mice increased between 4 and 8 weeks of age; dwarf mice had very low serum IGF-I levels. In both normals and dwarfs, serum IGF-II levels were highest soon after birth and dropped steadily thereafter. Western ligand blots of serum IGFBPs with 125I-IGF-II as tracer revealed the expected bands 41·5, 38·5, 30–32 and 24 kDa. In normal mice the IGFBP-3 doublet was already detectable at 2 weeks of age, and its intensity increased with age. In dwarf mice the IGFBP-3 doublet was hardly detectable.

The changes of IGFs and their IGFBPs were studied in sera of dwarf mice after treatment with growth hormone (GH) and/or thyroxine (T4) for 4 weeks. In spite of a comparable growth response obtained using these hormones, serum IGF-I was increased only by GH treatment; a small but significant decrease of serum IGF-II was obtained following GH or T4 treatment. An increase of the IGFBP-3 doublet was only obtained with GH; T4 and GH+T4 had no effect. The rise of IGFBP-3 after GH treatment was accompanied by the formation of the IGFBP 150 kDa complex, as measured by neutral gel chromatography. The size distribution of 125 I-IGF-II was restored to normal, while with 125I-IGF-I only a small peak at 150 kDa was observed. Elution profiles of sera after treatment with T4 or GH+T4 were identical to those of dwarf controls.

The presence of the IGFBPs was investigated in media conditioned by liver and lung explants of normal and dwarf animals. In culture media of liver explants from normal mice, bands at 30–32 and 24 kDa predominated; the intensity of the IGFBP-3 doublet was relatively low. In dwarfs the 30–32 kDa predominated. In culture media of the lung from normal mice the IGFBP-3 doublet and the 24 kDa band were clearly visible; in dwarf mice IGFBPs could not be detected. We were unable to identify the 150 kDa IGFBP-complex in this medium using the size distribution of 125I-IGFs on neutral gel chromatography after incubation with the conditioned media. This was in contrast to data obtained with normal serum.

Our data suggest that serum IGFBP-3 and IGF-I are regulated by GH and not by T4. In dwarf Snell mice, serum IGF-II is down regulated by GH as well as T4. The 150 kDa IGFBP complex is absent in dwarfs and, when induced by GH, seems to have a high affinity for IGF-II.

Journal of Endocrinology (1994) 143, 191–198
Genistein but not staurosporine can inhibit the mitogenic signal evoked by lithium in rat thyroid cells (FRTL-5)
Authors: Takano T , Takada K , Tada H , Nishiyama S , Amino N
Published: December 2008

Long-term administration of lithium is one of the well-known causes of goiter. It can stimulate DNA synthesis in rat thyroid cells (FRTL-5) treated with thyroid-stimulating hormone (TSH). To investigate the mitogenic signal transduction system activated by lithium, lithium-induced DNA synthesis and Ca2+ influx were studied using two protein kinase inhibitors, genistein as a specific tyrosine kinase inhibitor and staurosporine as a potent inhibitor of protein kinase C.

Genistein but not staurosporine blocked the DNA synthesis induced by lithium in TSH-primed cells but neither compound had any effect on the Ca2+ entry stimulated by lithium. Genistein clearly attenuated the phosphotyrosine content of the 175 kDa substrate in the presence of lithium but staurosporine failed to do so. Moreover, lithium could also stimulate DNA synthesis in protein kinase C down-regulated cells.

These data demonstrate that lithium may require the activation of a particular genistein-sensitive kinase, possibly a tyrosine kinase, to induce cell proliferation. It is suggested that the phorbol ester-sensitive protein kinase C family might not participate in the mitogenic signal transduction pathway activated by lithium.

Journal of Endocrinology (1994) 143, 221–226
Studies on the use of cultured cells in a bioassay for parathyroid hormone
Authors: Armston A E , Wood P J
Published: December 2008

Measurement of parathyroid hormone (PTH) is important for diagnosing hyper- and hypoparathyroidism. The move to two-site immunometric assays that detect the whole molecule has improved the discrimination of these conditions but these assays may be too restrictive because some PTH fragments that are biologically active may not be detected. In addition, PTH-like peptide of malignancy, an important cause of malignancy-associated hypercalcaemia, is not detected by the two-site assays.

Experiments were performed to set up a simple, robust and inexpensive bioassay for PTH, exploiting a kidney cell line and using cyclic AMP or an eluted stain assay as the end point. Of the 12 cell lines tested, an opossum kidney (WOK) cell line showed the most promise. Despite optimization of the procedure to include pre-treatment with dexamethasone, insulin and PTH, followed by incubation in the presence of 5′ -guanylimidodiphosphate, isobutyl-1-methylxanthine and forskolin, the WOK cells showed insufficient sensitivity for use in a cultured cell bioassay for PTH in human serum. In addition, the cells were less sensitive to PTH-like peptide precluding their use for an assay for this molecule.

Journal of Endocrinology (1994) 143, 261–268
Involvement of epidermal growth factor (EGF)/EGF receptor autocrine and paracrine mechanism in human trophoblast cells: functional differentiation in vitro
Authors: Amemiya K , Kurachi H , Adachi H , Morishige K-I , Adachi K , Imai T , Miyake A
Published: December 2008

We have studied the expression of epidermal growth factor (EGF) and EGF receptors (EGF-R) in isolated human trophoblast cells at various stages of differentiation and also the biological significance of the EGF/EGF-R autocrine and paracrine mechanism. Cytotrophoblast cells were isolated from human placental tissues of 6–9 weeks of gestation. Trophoblast cells underwent morphological and functional differentiation during in vitro culture. The expression of EGF and EGF-R protein and mRNA was studied in trophoblast cells cultured for 0–5 days, using immunocytochemical staining, and reverse transcription and polymerase chain reaction. Monoclonal antibodies (mAbs) against EGF and EGF-R showed specific staining in trophoblast cells at all stages of differentiation. Both EGF and EGF-R gene transcripts were detected in RNA samples isolated from trophoblast cells at all stages. These data suggest the presence of an EGF/EGF-R autocrine and paracrine mechanism in human trophoblast cells. Next, we examined the biological significance of this mechanism on trophoblast cell differentiation in vitro. EGF added to the culture medium significantly increased human chorionic gonadotrophin-β (hCG-β) secretion and, more importantly, anti-EGF neutralizing mAbs significantly reduced both hCG-β and human placental lactogen secretion from trophoblast cells in culture. All these results suggest that human trophoblast cells express both EGF and EGF-R, and that EGF may play an important role in the functional differentiation of human trophoblast cells.

Journal of Endocrinology (1994) 143, 291–301
Endothelin-1 is involved in the transient hypoparathyroidism seen in patients undergoing thyroid surgery
Authors: Nomura K , Yamashita J , Ogawa M
Published: December 2008

We have studied the serial changes in plasma endothelin (ET)-1, serum parathyroid hormone (PTH) and serum calcium concentrations during surgery in 20 patients undergoing a hemithyroidectomy for thyroid carcinoma. The plasma ET-1 concentrations increased within the first 24 h following surgery in 18 out of 20 cases with the peak concentration achieved between 1 and 12 h. Gel permeation chromatography showed only one major peak at the elution position of human ET-1 standard both in the pre- and postoperative plasma. Thirteen patients showed transient hypoPTHaemia and hypocalcaemia which clearly coincided with the peak plasma ET-1 concentration in 12. However, there was no significant association between the extent of the increase in the plasma ET-1 levels and the incidence of the transient hypoparathyroidism. When blood was sampled at multiple sites during surgery, the plasma ET-1 concentrations tended to be higher in the internal jugular vein on the side of the preserved thyroid lobe (4·92 ±2·21 pg/ml) as well as on the side of the lobectomy (4·36 ±1·95 compared with that in the antecubital vein (3·66± 1·44). Furthermore, tissue extracts from the parathyroid and thyroid contained considerably greater amounts of immunoreactive (ir-) ET-1 than most of the other tissues tested. In vivo experiments in animals revealed that a continuous infusion of ET-1 directly into the arterial bed supplying the preserved parathyroid glands can lead to a fall in serum PTH and Ca concentrations, whereas a bolus infusion into the artery or even a continuous infusion into the peripheral vein failed to do so, suggesting that the local increase in ET-1 may provoke this effect. All these results indicate that the transient increase in the plasma ET-1 level may be responsible for the transient hypoparathyroidism seen in patients after thyroid surgery despite successful preservation of the parathyroid glands. This effect may be mediated either through its vasoconstrictive activity or by its inhibitory effect on PTH secretion.

Journal of Endocrinology (1994) 143, 343–351
The effect of epitestosterone on gonadotrophin synthesis and secretion
Authors: Lapcik O , Perheentupa A , Bicikova M , Huhtaniemi I , Hampl R , Starka L
Published: December 2008

The effects of 3-week treatment with increasing doses of epitestosterone (ET) on gonadotrophin gene expression and secretion, on testosterone and 5α-dihydrotestosterone (DHT) levels, and on the weight of testes and prostates, were studied in intact adult male rats. The hormones were delivered by means of silastic capsules of different lengths filled with the steroid. One group of rats received testosterone (T) instead of ET, to compare the results with previous studies concerning the testosterone effect. The controls were given capsules with glucose only. Treatment with ET, as well as with T, significantly reduced the weights of prostates. When the data from ET-treated rats and controls were combined, a significant negative correlation (P<0·001) was found between the weight of prostates and serum ET. T, in contrast to ET, also decreased significantly the weights of testes. ET treatment caused a significant reduction of serum T levels but only an insignificant decline of DHT levels, independent of the dose. Serum and pituitary (p) luteinizing hormone (LH) levels in the ET-treated rats did not change. Pituitary mRNA contents for the βLH subunit (βLH-mRNA) showed a dose-dependent significant increase, up to 170% (P<0·01), with ET treatment. pFSH decreased with the lowest ET (2 cm) dose (P<0·05), but no change was observed with the other doses. The mRNA for the common α-subunit also increased with the ET load. In conclusion, ET acts at several sites in the regulation of gonadotrophin formation and release. It enhances the steady-state mRNA levels of both gonadotrophins in the pituitary. At the same time, ET may act directly in the pituitary by inhibition of post-transcriptional events in LH synthesis. A direct inhibitory effect of ET at the hypothalamic level is also possible. The circulating levels of both gonadotrophins are thus the result of these composite effects.

Journal of Endocrinology (1994) 143, 353–358
Influence of tumour necrosis factor-α, tumour necrosis factor-β and interferon-γ, separately and added together with interleukin-1 β, on the function of cultured human thyroid cells
Authors: Rasmussen Å K , Kayser L , Feldt-Rasmussen U , Bendtzen K
Published: December 2008

Interleukin (IL)-1, tumour necrosis factor (TNF)-α and interferon (IFN)-γ have been demonstrated in thyroid tissue. We have previously shown that high concentrations of IL-1 inhibit and low concentrations stimulate human thyroid cell function in vitro. In the present study, TNF-α, TNF-β and IFN-γ all inhibited thyroglobulin (Tg) and cAMP production from cultured human thyroid cells. When TNF-α was added simultaneously with IL-1 β, the highest concentration of TNF-α (106 U/l) enhanced the inhibition of Tg and cAMP induced by IL-1β (1–105 U/1). TNF-β had no influence on IL-1β-induced inhibition. IFN-γ (104 U/1) added together with IL-1β in lower concentrations (1—102 U/l) stimulated cAMP production, while at high concentrations of IL-1β (105 U/1), IFN-γ enhanced the inhibitory influence of IL-1 β on Tg production.

The hormones of the immune system, IL-1, TNF and IFN-γ, may thus contribute to the decreased thyroid function characteristic of some thyroid inflammatory diseases.

Journal of Endocrinology (1994) 143, 359–365
Differential response of neuropeptide Y, substance P and vasoactive intestinal polypeptide in the rat anterior pituitary gland to alterations in thyroid hormone status
Authors: Jones P M , Ghatei M A , Wallis S C , Bloom S R
Published: December 2008

We have compared the effects of thyroidectomy with those of thyroxine (T4) replacement and excess T4 treatment on neuropeptide Y (NPY) in the rat anterior pituitary, and compared these with the effects on substance P (SP) and vasoactive intestinal peptide (VIP). Thyroidectomy produced large increases in the peptide content of NPY (335 ± 58 fmol/gland vs 15 ± 4 fmol/gland in controls), SP (581 ±90 vs 199 ±32 fmol/gland) and VIP (1386 ± 395 vs 417 ± 77 fmol/gland) together with large increases in the respective prohormone encoding mRNAs, NPY 21 760%±1290%, preprotachykinin-A (PPT-A; which encodes the substance P precursor) 1744%± 190% and VIP 680% ± 129%. Thyroidectomy together with T4 replacement produced an increase in both NPY peptide (426 ±72 vs 15 ±4 fmol/gland) and mRNA content 970%±156% of controls). The peptide contents of SP and VIP were not significantly different from controls. PPT-A and VIP mRNA levels were decreased relative to controls (31%±8% and 23%± 10% respectively). In intact animals treated with excess T4 (hyperthyroid animals), SP and VIP peptide contents were significantly reduced (55 ±13 vs 199±32 fmol/gland and 226 ± 24 vs 417± fmol/gland respectively) and the SP and VIP encoding mRNAs were also decreased (8% ±3% and 11%±4% respectively). In this group there was no detectable alteration in either the peptide or mRNA content of NPY. Thus, the response of pituitary NPY to thyroid hormone manipulations cannot be explained in terms of negative feedback physiology and is different from those of SP and VIP. The results suggest that the regulation of locally produced NPY in the rat anterior pituitary is complex and may be influenced by thyroidal factors in addition to T4.

Journal of Endocrinology (1994) 143, 393–397
High plasma insulin-like growth factor-II and low lipid content in transgenic mice: measurements of lipid metabolism
Authors: Da Costa T H M , Williamson D H , Ward A , Bates P , Fisher R , Richardson L , Hill D J , Robinson I C A F , Graham C F
Published: December 2008

Transgenic mice were made by introducing extra copies of the mouse insulin-like growth factor-II (IGF-II) gene driven by the bovine keratin 10 promoter (BKVI). The adult plasma IGF-II levels were elevated at least three times in one line. In this line, there was a lower lipid content of both brown and white adipose depots at 2–4 months of age, and 40% less fat in the carcass at 7–9 months. The low lipid phenotype was not detected in the carcass at 2 weeks after birth. The lean characteristic was attributed to circulating IGF-II because the transgene was not expressed in fat. At 2–4 months of age, the transgenes oxidized more oral lipid, and less of this lipid was incorporated into the whole body and the epididymal fat. In contrast, the interscapular brown adipose tissue maintained lipid incorporation and lipoprotein lipase activity despite its reduced size. The altered activity of the brown adipose tissue may account for the gradual onset and persistence of the lean feature of the transgenic mice. There were no substantial changes in lipogenesis which could account for the low fat content. The plasma levels of IGF-I, insulin, glycerol, non-esterified fatty acids, triacylglycerols and glucose were not greatly changed and the pituitary GH content was within the normal range.

Journal of Endocrinology (1994) 143, 433–439
Pituitary-specific transcription factor Pit-1 in the rdw rat with growth hormone- and prolactin-deficient dwarfism
Authors: Ono M , Harigai T , Furudate S
Published: December 2008

The pituitary gland of the rdw rat (gene symbol: rdw) with hereditary dwarfism expresses 30–100 times less GH and prolactin (PRL) mRNA than normal controls. To clarify the features of rdw rats, TSH and the pituitary-specific transcription factor Pit-1, which is involved not only in the gene expression of GH and PRL but in somatotroph, lactotroph and thyrotroph development as well, were examined. The rdw pituitary contained about seven times more TSHβ mRNA than the normal control, whereas Pit-1 mRNA expression in rdw and control was the same. Nucleotide sequencing of PCR-amplified Pit-1 cDNA indicated that the deduced amino acid sequence of rdw Pit-1 was identical with that of the normal rat. Using an antibody against rat Pit-1 protein produced in E. coli, Western blotting analysis demonstrated the presence of the same amount of Pit-1 protein in rdw and normal rat pituitaries. The distribution of Pit-1-positive cells in the anterior pituitary was essentially the same in rdw and normal rats. It follows from these findings that the defective gene in the rdw rat is unrelated to the Pit-1 gene and the normal quantity of Pit-1 protein is insufficient to produce normal amounts of GH and PRL in the rdw pituitary. These and previous results suggest that the reduction in GH and PRL production in the rdw pituitary might be due to that in thyroid hormone production.

Journal of Endocrinology (1994) 143, 479–487
ANF(1-28) is a Potent Suppressor of Pro-opiomelanocortin (POMC) mRNA but a Weak Inhibitor of βEP-LI Release from AtT-20 Cells
Authors: Tan Tean T , Yang Zhiyu , Huang Weiqing , Lim Alan T.
Published: December 2008

Controversies remain whether atrial natriuretic factor (ANF) may play a role in modulating the release of POMC derived peptides from pituitary corticotrophs. Employing AtT-20 mouse pituitary tumour cells, we report here the effects of rat ANF(1–28) and sodium nitroprusside (SNP), both of which augment cellular levels of cGMP through activating particulate and soluble guanylyl cyclases respectively, on the expression of POMC mRNA abundance. Furthermore, the cellular contents and secretion of (beta endorphin-like immunoreactivity) βEP-LI from these cultures were also examined. Whereas the abundance of POMC mRNA was found to be markedly suppressed following 4h of incubation with rANP(1–28) (0.01 to 1 μM), SNP (0.1 to 10 μM) and dibutyryl-cGMP (1 to 100 μM) in a dose related manner, only a modest reduction in the release and cell contents of βEP-LI was found in some of these cultures. It is also of interest to note that in all the cases examined, the inhibitory effect was associated with a significant suppression of cAMP levels in the cultures. Taken together, our present findings suggest that ANF may play a more important role in suppressing the production than the release of POMC related peptides from AtT-20 cells. Thus, it raises the possibility that hypothalamic ANF may likewise modulate the function of the pituitary-adrenal axis through exerting a greater effect on inhibiting the production than the secretion of pituitary ACTH.
Influence of progesterone on boar sperm capacitation
Authors: Barboni B , Mattioli M , Seren E
Published: December 2008

This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITCconjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation.

Journal of Endocrinology (1995) 144, 13–18
Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours
Authors: Stridsberg M , Öberg K , Li Q , Engström U , Lundqvist G
Published: December 2008

Chromogranins and/or secretogranins constitute a family of water-soluble acidic glycoproteins that are present in almost all endocrine, neuroendocrine and neuronal tissue. Antibodies against chromogranins have been widely used for immunohistochemical staining of endocrine tissue and tumours of neuroendocrine origin. Furthermore, measurements of circulating chromogranin A have been used as a reliable marker for neuroendocrine tumour growth. In this study, we describe the development of specific antibodies against chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin. The antibodies were used for immunohistochemical staining of normal and neoplastic neuroendocrine tissue and development of reliable radioimmunoassays for chromogranin A, chromogranin B, chromogranin C and pancreastatin. In 44 patients with carcinoid tumours, 17 patients with sporadic endocrine pancreatic tumours and 11 patients with endocrine pancreatic tumours and the multiple endocrine neoplasia 1 syndrome, plasma measurements revealed elevated chromogranin A levels in 99%, elevated chromogranin B in 88%, elevated chromogranin C in 6% and elevated pancreastatin in 46% of the patients. Urinary measurements revealed elevated levels in 39%, 15%, 14% and 33% of the patients respectively. Gel permeation chromatography of plasma and urine showed that circulating chromogranin A, and immunoreactive fragments of chromogranin A, had a higher molecular weight distribution than the chromogranin A fragments excreted to the urine. Furthermore, it was noted that most of the patients excreting chromogranin A fragments to the urine had previously been treated with streptozotocin, a cytotoxic agent known to induce renal tubular dysfunction. The antibodies raised proved useful for immunohistochemical staining and visualised endocrine cells in pancreatic islets, adrenal medulla and the small intestine as well as in endocrine pancreatic tumours, pheochromocytoma and midgut carcinoid tumours. In conclusion, the antibodies raised were useful for both immunohistochemical staining of normal tissue and endocrine tumours as well as development of specific radioimmunoassays for plasma measurements of the different chromogranins. Furthermore, we show that plasma measurements of chromogranin A and B were superior to measurements of chromogranin C and pancreastatin and plasma measurements of the different chromogranins were more reliable as markers for tumour growth than the corresponding urine measurements.

Journal of Endocrinology (1995) 144, 49–59
A homologous radioimmunoassay for ovine insulin-like growth factor-binding protein-2: ontogenesis and the response to growth hormone, placental lactogen and insulin-like growth factor-I treatment in sheep
Authors: Gallaher B W , Breier B H , Blum W F , McCutcheon S N , Gluckman P D
Published: December 2008

Although insulin-like growth factor-binding protein-2 (IGFBP-2) is an abundant IGFBP in fetal and postnatal plasma, its regulation is not yet clearly understood. To address this question in sheep, we purified ovine IGFBP-2 and developed a homologous radioimmunoassay. We have studied its ontogenesis and measured serum concentrations of ovine IGFBP-2 after bovine growth hormone (bGH), ovine placental lactogen (oPL) and IGF-I treatment.

Concentrations of IGFBP-2 were high at 125 days of gestation (550 ± 15 μg/l) but fell after birth P<0·05) and plateaued after 1 year of age (340 ± 20 μg/l). In lactating ewes, bGH treatment for 7 days significantly reduced (21%; P<0·05) IGFBP-2 relative to the saline-treated group. Similarly, in neonatal lambs, bGH treatment from day 3 to day 23 of life reduced (P<0·05) IGFBP-2 by 23% relative to the saline-treated group. oPL had no effect on serum levels of IGFBP-2 in the ewe or the neonatal lamb. In well-fed yearling lambs, treatment with IGF-I reduced IGFBP-2 values by 27% (P<0·05) relative to control animals. In yearling lambs, reduced nutrition increased plasma IGFBP-2 (41%; P<0·05). However this increase was abolished by IGF-I treatment.

The changes in plasma levels of IGFBP-2 were positively related to changes in IGF-II while there was a negative relationship between circulating IGF-I and IGFBP-2 such that both IGF-I and IGF-II may play a role in the regulation of IGFBP-2 in serum.

Journal of Endocrinology (1995) 144, 75–82
Changes induced by non-enzymatic glycosylation of IGF-binding protein-3: effects on its binding properties and on its modulatory effect on IGF-I mitogenic action
Authors: Cortizo A M , Gagliardino J J
Published: December 2008

The aim of this study was to demonstrate the feasibility of in vitro non-enzymatic glycosylation of IGF-binding protein-3 (IGFBP-3) and whether this process affects its binding properties and its modulatory effect on IGF-I mitogenic activity. Swiss 3T3 fibroblasts were cultured and the IGFBP-3 released into the medium (CM) glycated with either labelled or unlabelled glucose. Parallel glycation studies were performed using standard human IGFBP-3. Both species of IGFBP-3 became effectively glycated in a dose-dependent manner. Glycated IGFBP-3 bound larger amounts of 125I-labelled IGF-I than its non-glycated form. According to Scatchard analysis this effect might be due to an increase in the number of binding sites of the IGFBP-3 molecule rather than to changes in its affinity constants, which remain unchanged. Preincubation of fibroblasts with CM containing IGFBP-3 for 16 h before the addition of IGF-I enhanced the stimulatory effect of the hormone on thymidine incorporation into cell DNA. This potentiation was blunted when in vitro glycated instead of non-glycated IGFBP-3 was employed. These results provide further evidence of the in vitro glycation of IGFBP-3 and demonstrate that this process affects both its binding properties and its enhancing effect on IGF-I mitogenic activity. These changes may explain, at least partially, the development of many alterations observed in poorly controlled diabetic patients.

Journal of Endocrinology (1995) 144, 119–126
ETA and ETB endothelin receptors in human myometrium characterized by the subtype selective ligands BQ123, BQ3020, FR139317 and PD151242
Authors: Bacon C R , Morrison J J , O'Reilly G , Cameron I T , Davenport A P
Published: December 2008

ETA selective (BQ123, FR139317, PD151242) and ETB selective (BQ3020) ligands were used to define the binding characteristics and contractile function of endothelin receptor subtypes in human myometrium. In saturation binding assays with 10 μm-thick tissue sections [125I]endothelin-1 (ET-1) bound with a single affinity to receptors in the myometrium (Kd, 1·19±0·17 nm) and adjacent endometrium (Kd, 1·39±0·51 nm). Competition binding assays in myometrium revealed a heterogeneous population of receptors with BQ123 (Kd ETA, 1·43±0·33 nm; Kd ETB, 39·91±9·06 μm), FR139317 (Kd ETA, 2·54±0·87 nm; Kd ETB, 89·79±24·34 μm) and BQ3020 (Kd ETA, 4·57±0·58 μm; Kd ETB, 90·07±19·53 nm). The presence of these receptors in myometrium was confirmed by saturation assays with the new ETA selective ligand [125I]PD151242 (Kd, 0·93±0·08 nm Bmax 138·7±1·0 fmol/mg protein) and the ETB selective [125I]BQ3020 (Kd, 0·62±0·07; Bmax 44·5±1·1 fmol/mg protein). Reverse-transcriptase PCR assays detected mRNA encoding both receptor subtypes in myometrium.

Autoradiography with radiolabelled PD151242 and BQ3020 demonstrated that ETA receptors were the predominant subtype in the myometrium and identified a population of ETB receptors in the endometrium.

In tissue bath experiments, an ET-1-induced increase in contractility of myometrial strips was antagonized by 10 μm FR139317 but not by BQ123 at the same concentration. The ETB agonist BQ3020, which is a potent agonist in animal tissue, did not increase contractility when tested at concentrations up to 2 μm.

The results of this study suggest that ETA endothelin receptors and a smaller population of ETB receptors are found in human myometrium. ETA, but not ETB, receptors mediate myometrial contractility. The function of ETB receptors in human uterus remains to be elucidated.

Journal of Endocrinology (1995) 144, 127–134
Evidence for chromium acting as an essential trace element in insulin-dependent glucose uptake in cultured mouse myotubes
Authors: Morris B , Gray T , MacNeil S
Published: December 2008

Previous work from this and other laboratories has suggested that the trace element chromium plays some role in glucose homeostasis. In this study, we sought to characterise an in vitro cell culture model in which the effects of chromium on insulin-dependent glucose uptake could be studied. Mouse C2C12 myoblasts were differentiated to form myotubes in culture in chromium-replete or chromium-poor media. Chromium levels in standard media were 0·56±0·01 μg/l (mean ± s.e.m.) compared with 0·09±0·01 μg/l in chromium-poor media.

In chromium-poor media, insulin-stimulated uptake of radiolabelled glucose was reduced by almost 50% compared with that found in chromium-replete media. This decreased response could be restored by the addition of physiologically relevant (0·3 μg/l) concentrations of inorganic chromium (P<0·001). The sensitivity of these cells to insulin was reduced dramatically by a reduction in the chromium content of the medium and was again increased (P<0·001) by chromium addition. The concentrations of chromium required to restore the sensitivity to insulin were of the same order as those found physiologically; much higher concentrations of chromium could also stimulate glucose uptake in the absence of insulin but such concentrations were supra-physiological.

Journal of Endocrinology (1995) 144, 135–141
Transforming growth factor-α: receptor binding and action on DNA synthesis in the sheep mammary gland
Authors: Moorby C D , Taylor J A , Forsyth I A
Published: December 2008

Microsome fractions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-α (TGF-α). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-α and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (Kd) was similar in all physiological states (2·43±0·27 mol/l × 10−10, n=23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14·07± 2·45 fmol/mg protein, n=10) than in non-pregnant, 10-or 15-week pregnant sheep (43·04±5·93 fmol/mg protein, n=13).

DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-a (maximum response at 10 μg/l; 1·8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-α on day 3 of culture, but the response was delayed to day 4–5 of culture in cells from other physiological states. Dose–response was not significantly affected. TGF-α and IGF-I produced an additive effect on DNA synthesis. Oestradiol (10−12 to 10−9 m), a potential stimulator of the TGF-α gene, did not stimulate DNA synthesis alone, or in combination with IGF-I. It is concluded that growth factors acting via the EGF receptor play a role in ruminant mammary development, but whether they mediate oestradiol effects remains unresolved.

Journal of Endocrinology (1995) 144, 165–171
Scanning electron microscope assessment of exocytotic changes in purified gonadotropes
Authors: Mizuki J , Masumoto N , Tahara M , Fukami K , Mammoto A , Tasaka K , Miyake A
Published: December 2008

These studies were undertaken to characterize the exocytotic changes in purified gonadotropes by three-dimensional imaging using scanning electron microscopy. Rat gonadotropes were purified using a fluorescence-activated cell sorter and an argon laser treatment system. The purified gonadotropes were stimulated with GnRH under various conditions and fixed for scanning electron microscopy. After the GnRH stimulation, many 'hole' structures (diameter 0·1–0·5 μm) were observed on the cell surface, and notably the population of cells with 10 or more holes was clearly increased. The pattern of the time-course of the changes in this population was perfectly consistent with the LH secretory profile of pituitary cells, and their formation of the cells with 10 or more holes was completely inhibited by pretreatment with a GnRH antagonist. Our data suggest that the hole structure represents an exocytotic opening site and that regulated exocytosis in purified gonadotropes can be evaluated by scanning electron microscopy. This method may be widely applicable to other endocrine cells.

Journal of Endocrinology (1995) 144, 193–200
Inositol 1,4,5-trisphosphate 3-kinase activity in FRTL-5 cells: regulation of the enzyme activity by TSH
Authors: Takazawa K , Go M , Endo T , Erneux C , Onaya T
Published: December 2008

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase phosphorylates the Ca2+-mobilizing second messenger InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 5-phosphatase dephosphorylates InsP3 to inositol 1,4-bisphosphate (InsP2). We compared the effects of TSH added to a culture of FRTL-5 thyroid cells on the activity of InsP3 5-phosphatase and InsP3 3-kinase. InsP3 3-kinase activity was decreased at a physiological concentration of TSH. Inhibition of this activity started after 3 h of incubation with TSH and was maximal after 24 h. In contrast, InsP3 5-phosphatase activity was not affected by TSH under the same conditions. The inhibitory effect of TSH on InsP3 3-kinase was characterized as follows: a) inhibition of activity was mimicked by both dibutyryl cyclic AMP and forskolin; b) activity obtained by mixing lysates of TSH-stimulated and non-stimulated cells was the sum of each activity measured separately; c) inhibition persisted after a crude lysate of TSH-stimulated cells had been subjected to SDS/polyacrylamide gel electrophoresis and the extraction of InsP3 3-kinase activity. The data suggest that TSH reduced the activity of InsP3 3-kinase in FRTL-5 cells either by a phosphorylation/dephosphorylation mechanism, or by affecting expression of the enzyme.

Journal of Endocrinology (1995) 144, 527–532
Anti-progesterone antibody administration and the impairment of postpartum maternal care in mice
Authors: Wang M-W , Crombie D L , Mais D E , Hayes J S , Heap R B
Published: December 2008

Passive transfer of a monoclonal antibody against progesterone produces a high incidence of maternal rejection in mice after recovery from antibody-induced infertility. To investigate the mechanisms involved in this reduction of maternal care, we have examined whether the effect is due to long-term exposure to antibody. Antibody was administered i.p. either on day 2 or day 17 of pregnancy. When a low dose (1·0 nmol) was given on day 2, pregnancy proceeded normally but 44·8% pups delivered at term were rejected compared with 12·7% in the control group. When a higher dose (4·5 nmol) of antibody was given on day 17, pregnancy continued normally to term and the rejection rate was 48·8% (control: 11·1%). When the same amount of antibody was injected after delivery (day 1 of lactation), no detrimental effect was found on subsequent maternal care to the young, the rejection rate being comparable between antibody-treated and control groups (5·3% vs 4·6%).

To determine if the presence of antibody interfered with lactation or suckling, a bolus injection of 10 μCi [3H]H2O was given to mice treated at day 17 with antibody or saline. The levels of radioactivity present in both mothers and pups and the first 5-day pup growth curves showed identical patterns, indicating that milk availability and the suckling process were not affected. Crossfostering studies revealed that antibody-treated mothers rejected 25·5% of fostered pups compared with 8·5% found in the control females when antibody was administered on day 17 of pregnancy and the entire litters were crossfostered between the two groups immediately after delivery. Detailed analyses using a retrieval test further demonstrated that the reduction in maternal care was most pronounced during the first 3 days after delivery.

These results demonstrate that antibody-treated mothers show a higher frequency of pup rejection which is (i) not restricted to their own litter; (ii) not due to lack of milk; and (iii) not a result of defects in the pups. They are consistent with the hypothesis that anti-progesterone antibody interferes with the priming mechanism(s) necessary for the onset of maternal behaviour during the 3-day period prior to delivery, leading to impaired maternal care after parturition.

Journal of Endocrinology (1995) 145, 363–369
Aberrant maternal behaviour in mice treated with a progesterone receptor antagonist during pregnancy
Authors: Wang M-W , Crombie D L , Hayes J S , Heap R B
Published: December 2008

The rapid onset of normal maternal behaviour after parturition in mice, consisting of cleaning, warming, feeding and protection of offspring, is primed by oestrogen, progesterone and oxytocin. Previous studies showed that passive transfer of monoclonal antibodies against progesterone significantly increases the incidence of maternal rejection of pups. To test the hypothesis that aberrant maternal behaviour is due to partial progesterone withdrawal leading to hormonal imbalance during late pregnancy, maternal rejection was assessed following treatment with a progesterone receptor antagonist. Mifepristone (RU486) was given subcutaneously on either day 2 (100 μg) or day 17 (50 μg) of pregnancy, or on the first day of lactation (100 μg). Maternal behaviour was monitored twice daily for the first 6 days of lactation and pup rejection recorded for a further 15 days. Maternal rejection was significantly greater after mifepristone administration on either day 2 or day 17 (28·6% and 38·3%) compared with controls (11·1% and 5·2% respectively). Rejection was negligible in both treated and control groups if mifepristone was given after parturition. When mothers were treated at day 17, the length of the latent period before pups were retrieved and returned to the nest was markedly increased in mifepristone-treated mothers (46·3 s) compared with controls (4·4 s) though the effect was transient. The results indicate that mifepristone interferes with the hormonal priming mechanism(s) necessary for the onset of normal maternal behaviour by a receptor-mediated effect. The similarity of the present results and those obtained with anti-progesterone antibodies implies that receptor antagonism or antibody scavenging of progesterone influence a common central nervous mechanism that is essential for the normal priming process.

Journal of Endocrinology (1995) 145, 371–377
Effects of undernutrition and diabetes on serum and liver mRNA expression of IGFs and their binding proteins during rat development
Authors: Rivero F , Goya L , Aláez C , Pascual-Leone A M
Published: December 2008

The purpose of the present study was to investigate the influence of nutrients and insulin on IGFs and their binding proteins (IGFBPs) during the fetal and neonatal periods of three rat populations: (a) rats undernourished by a 35% reduction in the diet from day 16 of gestation, (b) streptozotocin-induced diabetic rats from the same day, or 4 days after birth, and (c) control rats. Fetuses from the diabetic population showed a decrease in insulinemia at 19 and 21 days, along with an increase in glycemia at all stages. Neither glycemia nor insulinemia changed in the fetuses of undernourished mothers, but body weight was decreased at birth. Serum IGF-II decreased at 18 and 19 days of gestation in fetuses from undernourished mothers, and increased at 18, 19 and 21 days in fetuses from diabetic mothers. Serum IGFBPs of low molecular weight (IGFBP-1 and IGFBP-2) increased in the three fetal populations studied, although no changes in serum IGFBPs were found from the effect of undernutrition or diabetes, but fetal liver IGFBP-1 mRNA expression was found to be decreased in undernourished and diabetic animals as compared with controls. In neonatal rats, body weight, insulinemia and serum GH decreased in both undernourished and diabetic rats vs controls, while glycemia decreased in the undernourished and increased in the diabetic group. Serum IGF-II decreased only in diabetic rats and serum IGF-I decreased in both groups. The neonatal serum 30 kDa complex (IGFBP-1 and -2) also increased in undernutrition and diabetes parallel to the expression of mRNA. But, taken together, the changes in IGFBP peptide levels and liver mRNA expression strongly suggest that the 30 kDa complex seems to be composed mostly of IGFBP-1 in the diabetic group and of both IGFBP-1 and -2 in the undernourished animals. The studies of liver mRNA expression of IGFs and IGFBPs confirm the different metabolic control mechanism for the availability of IGFs by the IGFBPs, depending on the animal's maturity. The different adaptation shown by the diabetic neonatal population was confirmed by correlation studies between body weight, glycemia, insulinemia, IGF-I and IGFBPs. The different mechanism of adaptation in diabetic vs undernourished rats seems to be probably due to the decisive role played by hyperglycemia in the diabetic population, and also shows the crucial influence of nutritional status on IGFs and IGFBPs.

Journal of Endocrinology (1995) 145, 427–440