bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-like growth factor-I (IGF-I) production by bovine granulosa cells in vitro and peripheral IGF-I measurement in cattle serum: an evaluation of IGF-binding protein extraction protocols
Authors: Gutiérrez C G , Campbell B K , Armstrong D G , Webb R
Published: December 2004

Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.

As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.

It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

Journal of Endocrinology (1997) 153, 231–240
Possible role of corticosterone in the down-regulation of the hypothalamo-hypophysial-thyroid axis in streptozotocin-induced diabetes mellitus in rats
Authors: van Haasteren G A C , Sleddens-Linkels E , van Toor H , Klootwijk W , de Jong F H , Visser T J , de Greef W J
Published: December 2004

We investigated the effects of diabetes mellitus on the hypothalamo-hypophysial-thyroid axis in male (R×U) F1 and R-Amsterdam rats, which were found to respond to streptozotocin (STZ)-induced diabetes mellitus with no or marked increases, respectively, in plasma corticosterone. Males received STZ (65 mg/kg i.v.) or vehicle, and were killed 1, 2 or 3 weeks later. At all times studied, STZ-induced diabetes mellitus resulted in reduced plasma TSH, thyroxine (T4) and 3,5,3′-tri-iodothyronine (T3). Since the dialyzable T4 fraction increased after STZ, probably as a result of decreased T4-binding prealbumin, plasma free T4 was not altered during diabetes. In contrast, both free T3 and its dialyzable fraction decreased during diabetes, which was associated with an increase in T4-binding globulin. Hepatic activity of type I deiodinase decreased and T4 UDP-glucuronyltransferase increased after STZ treatment. Thus, the lowered plasma T3 during diabetes may be due to decreased hepatic T4 to T3 conversion.

Median eminence content of TRH increased after STZ, suggesting that hypothalamic TRH release is reduced during diabetes and that this is not caused by impaired synthesis or axonal transport of TRH to the median eminence. Hypothalamic proTRH mRNA did not change in diabetic (R×U) F1 rats during the period of observation, but was lower in R-Amsterdam rats 3 weeks after STZ. Similarly, pituitary TSH and TSHβ mRNA had decreased in R-Amsterdam rats by 1 week after STZ treatment, but did not change in (R×U) F1 rats. The difference between the responses in diabetic R-Amsterdam and (R×U) F1 rats may be explained on the basis of plasma corticosterone levels which increased in R-Amsterdam rats only. Hypothalamic TRH content was not affected by diabetes mellitus, but the hypothalami of diabetic rats released less TRH in vitro than those of control rats. Moreover, insulin had a positive effect on TRH release in vitro.

In conclusion, the reduced hypothalamic TRH release during diabetes is probably not caused by decreases in TRH synthesis or transport to the median eminence, but seems to be due to impaired TRH release from the median eminence which may be related to the lack of insulin. Inhibition of proTRH and TSHβ gene expression in diabetic R-Amsterdam rats is not a primary event but appears to be secondary to enhanced adrenal activity in these animals during diabetes.

Journal of Endocrinology (1997) 153, 259–267
Concentration of free growth hormone-binding protein in the serum of mice is not regulated by growth hormone
Authors: Sotelo A I , Dominici F P , Bartke A , Turyn D
Published: December 2004

Ames dwarf mice that do not express growth hormone (GH) or prolactin (PRL) genes were used to study the effects of GH deficiency on the presence and the characteristics of GH-binding protein (GHBP) in serum. Chromatographic techniques were used to allow characterization of biological rather than immunological activity of GHBP. Two GH-binding fractions were found in dwarf mice serum, one with low affinity and high capacity (GHBPI) and one with high affinity, low capacity and lower molecular mass (GHBPII). Serum concentration of the high-affinity GHBP was 0·73 ± 0·03 nm with a Kd of 6·3 ± 1·7 nm. Since Ames dwarf mice have no GH in the circulation, all the GHBP is free. Interestingly, the concentration of GHBP in dwarf mice was similar to the levels of free GHBP measured in normal mice from the same line. Moreover, this value (0·7 nm) closely resembles the concentration of free GHBP in the serum of transgenic mice overexpressing GH, in which peripheral GH levels are grossly elevated. These observations can be interpreted as evidence that the levels of free GHBP in mouse serum are independent of GH concentration, and that GH influences only the levels of bound GHBP in peripheral circulation.

Journal of Endocrinology (1997) 153, 319–325
Administration of IGF-I affects the GH axis and adolescent growth in normal monkeys
Authors: Wilson M E
Published: December 2004

The present study examined the effects of IGF-I on serum concentrations of IGF binding protein-3 (IGFBP-3) and GH and assessed how treatment with estradiol and IGF-I would stimulate adolescent growth in monkeys with normal pituitary function. In study I, ovariectomized, juvenile female rhesus monkeys (21 months of age; n=6) received a bolus injection of IGF-I (1 mg/kg s.c.) and serum samples were collected periodically through 48 h. The consequential elevation in serum IGF-I resulted in a parallel increase in serum IGFBP-3 at 1 and 3 h after treatment with values returning to baseline by 7 h. In contrast, the elevation in serum IGF-I resulted in a significant decline in serum GH within 3 h of treatment. These data confirm that an elevation in IGF-I increases IGFBP-3 while simultaneously acting in a negative feedback capacity to inhibit GH.

In study II, ovariectomized, juvenile female rhesus monkeys served either as controls (Con, n=6) or received a constant s.c. infusion of IGF-I (300 μg/day; Igf, n=6) from 13 through 32 months of age. At approximately 26 months, females entered an estradiol-treatment protocol in which they received alternating blocks of 3 weeks of estradiol followed by 3 weeks of no estradiol. As found in study I, the elevation in serum IGF-I resulted in a significant increase in serum IGFBP-3 throughout the study in Igf compared with Con females. Estradiol administration significantly increased serum IGF-I and IGFBP-3 levels in both groups. Although the nano-molar ratio of IGF-I to IGFBP-3 was consistently higher in Igf females, the magnitude of the change in IGF-I:IGFBP-3 following estradiol treatment was similar between groups. Finally, the age-dependent increase in serum GH was dampened in Igf compared with Con females and the increase in response to estradiol was less in Igf females. Although total growth in crown-rump length was similar in both groups, Igf females grew more prior to estradiol replacement while Con females grew more once estradiol treatment was initiated. In addition, skeletal maturity was advanced more quickly in Igf females once estradiol treatment had been initiated.

These data suggest that, in female monkeys with normal pituitary function, IGF-I administration inhibits endogenous GH secretion but is capable of stimulating crownrump growth. Although IGF-I increased serum levels of IGFBP-3, the increase was not proportional to the increase in serum IGF-I achieved by the treatment. These data would suggest that IGF-I may regulate the release of this binding protein but that GH may be required to maintain equi-molar proportions of IGF-I to IGFBP-3. In addition, the observation that serum concentrations of IGF-I were increased further in IGF-I-treated females by the administration of estradiol without a change in serum GH, suggests that estradiol has a direct effect on IGF-I synthesis and release independent of GH.

Journal of Endocrinology (1997) 153, 327–335
Prolactin receptors in human meningiomas: characterization and biological role
Authors: Muccioli G , Ghè C , Faccani G , Lanotte M , Forni M , Ciccarelli E
Published: December 2004

Sixty cerebral meningioma specimens obtained at surgery from 34 female and 26 male patients were examined for the presence of prolactin (PRL) receptors. These were compared with normal arachnoid tissue from which these tumours arise. PRL receptors were detected in 61·7% of meningiomas whereas no PRL binding was found in samples of normal arachnoid tissue. No relationship was found when sex or histological findings were compared with the presence of PRL receptors. Receptor-positive tumours had saturable and high-affinity (Kd, 4·8 ± 0·5 ng/ml) receptors with hormonal specificity for human PRL (hPRL) resembling that of other target tissues of PRL in man. The biological role of these receptors was investigated in primary cell cultures derived from meningioma tissue characterized for PRL receptor. When human PRL was added to the culture medium, in doses ranging from 1 to 200 ng/ml, a dose-dependent stimulation of 3H-thymidine incorporation was observed only in PRL-receptor positive tumours. The PRL concentrations required to produce a half-maximal effect ranged from 11 to 20 ng/ml and were quite close to the dissociation constant (Kd) of binding of PRL to its receptors. PRL also caused an increase of cell number compared with control with a significant effect after 3 and 4 days of culture.

In conclusion, these findings indicate that a large number of human meningiomas express specific and functional receptors for PRL which are involved in mediating its proliferative effects.

Journal of Endocrinology (1997) 153, 365–371
Effect of insulin-induced hypoglycaemia on secretion patterns and rates of corticotrophin-releasing hormone, arginine vasopressin and adrenocorticotrophin in horses
Authors: Alexander S L , Roud H K , Irvine C H G
Published: December 2004

To study the effect of hypoglycaemia on secretion rates of corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and ACTH in a non-ruminant species, a non-surgical method was used to collect pituitary venous (PitVen) blood every 0·5 or 1 min from seven horses before and after insulin administration (0·4 U/kg i.v.). To assess the effect of PitVen cannulation on results, peripheral hormones were also measured before and after insulin in five horses without PitVen cannulae.

Insulin administration lowered plasma glucose in all horses (P<0·0001; paired t-test). Cortisol concentrations, which were similar in horses with and without PitVen cannulae before insulin, rose significantly after insulin administration in both groups. Most horses showed discomfort as glucose fell. When data from horses with and without PitVen cannulae were pooled, the peak fractional change in cortisol (Spearman's rank correlation coefficient (rs)= −0·94, P<0·001) and the severity of hypoglycaemic symptoms (rs= −0·61, P<0·02) were inversely ranked with the glucose nadir. In horses with PitVen cannulae, insulin administration increased secretion rates of ACTH (P<0·0001), AVP (P<0·0001) and CRH (P<0·02). Increments in ACTH (rs=−0·96, P<0·005) and CRH (rs= −0·81, P<0·05), but not in AVP, measured during the second half-hour after insulin (i.e. the peak response), were inversely ranked with the glucose nadir. Moreover, ACTH increments were positively ranked with those in CRH (rs=0·81, P<0·05), but not in AVP. Nevertheless, in individual horses, minute-to-minute AVP and ACTH concentrations in PitVen blood were always correlated, whereas minute-to-minute CRH and ACTH concentrations were correlated only when glucose dropped below 3·4 mmol/l. In less hypoglycaemic horses, ACTH secretion rose despite little or no change in CRH.

We suggest that in horses AVP is the primary acute signal for ACTH release both before and during hypoglycaemia; however, the increasing magnitude of ACTH increments induced by greater degrees of hypoglycaemia is determined largely by selective CRH release, which then augments corticotroph responses to AVP.

Journal of Endocrinology (1997) 153, 401–409
Ca2+ receptor mRNA and protein increase in the rat parathyroid gland with advancing age
Authors: Autry C P , Kifor O , Brown E M , Fuller F H , Rogers K V , Halloran B P
Published: December 2004

Parathyroid hormone (PTH) release is regulated by extracellular calcium through a Ca2+ receptor (CaR) located on the surface of the parathyroid cell. With advancing age, the serum concentration of PTH increases, and evidence suggests that the calcium set-point for PTH release may also increase. To determine whether these changes are linked to a change in CaR expression, we quantitated mRNA and protein for the receptor in parathyroid glands of 6-week-, 6-month- and 24-month-old rats. Thyroid and kidney tissue were also studied. Between 6 weeks and 24 months of age, CaR mRNA in the parathyroid gland increased 11·4- and 3·3-fold as measured by competitive reverse transcription PCR and solution hybridization assays respectively. Message levels for the receptor also increased in the thyroid but not in the kidney. Coincident with the increase in message levels, receptor protein concentration in the parathyroid increased 7-fold between 6 weeks and 24 months of age. These results suggest that the altered relationship between extracellular calcium and PTH release observed in aging is associated with dramatic changes in CaR metabolism. That PTH secretion is increased despite increased receptor concentration suggests that aging may impair calcium binding or coupling between the CaR and down-stream effector elements in the pathway regulating PTH release.

Journal of Endocrinology (1997) 153, 437–444
Measurement of N- and C-terminal-region fragments of parathyroid hormone-related peptide in milk from lactating women and investigation of the relationship of their concentrations to calcium in milk
Authors: Uemura H , Yasui T , Yoneda N , Irahara M , Aono T
Published: December 2004

Parathyroid hormone-related peptide (PTHrP) is found in very high concentrations in the milk of various mammals. However, little is known about its physiological role in this fluid. To obtain detailed profiles of PTHrP in milk, we measured the concentrations of PTHrP in human milk by two different region-specific assays, PTHrP(1–87) (N-PTHrP) and PTHrP(109–141) (C-PTHrP). We also examined the correlations between PTHrP and Ca concentrations in milk as well as the correlations between PTHrP and secreted milk volume.

The levels of N-PTHrP and C-PTHrP were relatively low after delivery and gradually increased to 13·87 ± 2·40 nmol/l (mean ± s.e.m.) and 56·39 ± 11·31 nmol/l respectively on the 10th day postpartum. N-PTHrP concentration remained steady until the 6th month postpartum when weaning starts, at which point it decreased slightly. C-PTHrP levels changed in a similar way to N-PTHrP levels but were 2- to 5-fold higher. Milk Ca concentration, and content, correlated with C-PTHrP concentration, and content (r=0·422 and r=0·769 respectively; P<0·0001) but not with N-PTHrP. N-PTHrP concentration in the milk samples on the 4th day postpartum correlated with the volume of milk secreted during the 24 h before the samples were taken (r=0·524, P<0·01), but C-PTHrP concentration did not.

These results suggest that PTHrP in human milk may play some role in the maintenance of lactation through the N-terminal region and in promoting Ca transfer into milk via the C-terminal region.

Journal of Endocrinology (1997) 153, 445–451
Intracellular regulation of 17β-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells
Authors: Blomquist C H , Leung B S , Beaudoin C , Poirier D , Tremblay Y
Published: December 2004

There is growing evidence that various isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17β-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme–NAD or an enzyme–NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the Δ4 or Δ5 pathway is limited.

Journal of Endocrinology (1997) 153, 453–464
Modulation of human neutrophil function in vitro by gastrin
Authors: De la Fuente M , Carrasco M , Hernanz A
Published: December 2004

We have studied the effects in vitro of gastrin-17 and gastrin-34, at concentrations from 10−14 m to 10−6 m, on several of the functions of peripheral blood human neutrophils, i.e. adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), ingestion of inert particles (latex beads) and cells (Candida albicans) and superoxide anion production. Both gastrins inhibited several steps of the phagocytic process of human neutrophils, such as mobility and ingestion. By contrast, these peptides increased adherence and had no effect on superoxide anion production. In general, these effects were significant at peptide concentrations between 10−12 m and 10−8 m with a maximal effect at 10−10 m. In addition, gastrin peptides induced a significant increase in intracellular cAMP levels at 30, 60 and 120 s. Moreover, the inhibitory effect of gastrin-17 on the ingestion capacity of neutrophils (latex bead phagocytosis) was similar to that obtained with EGTA, a well-known extracellular calcium chelating compound. Gastrin-17 was found to inhibit completely the stimulation of latex bead phagocytosis in neutrophils caused by the calcium ionophore A23187. These results suggest that gastrin is a negative modulator of the phagocytic process of human neutrophils, and that this effect might involve an increase in intracellular cAMP levels and a decrease in calcium entry into the cells.

Journal of Endocrinology (1997) 153, 475–483
Immunolocalisation of oestrogen receptor-α within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood
Authors: Fisher J S , Millar M R , Majdic G , Saunders P T K , Fraser H M , Sharpe R M
Published: December 2004

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-α (ERα) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17·5 and 18·5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18–24, 54–62 and 92–112 weeks respectively. Immunolocalisation of ERa used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ERa was immunoexpressed in interstitial cells, including the fetal/neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ERa whereas the comparable cells in the marmoset were only weakly immunopositive. ERa was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ERa was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ERa. Apart from sporadic immunostaining for ERa in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ERa at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ERa, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ERβ) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Journal of Endocrinology (1997) 153, 485–495
The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA
Authors: Phaneuf S , Asbóth G , Carrasco M P , Europe-Finner G N , Saji F , Kimura T , Harris A , Bernal A López
Published: December 2004

We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 × 103 binding sites/cell, but this was time-dependently reduced to 27 × 103 sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.

Journal of Endocrinology (1997) 154, 7–18
Modulation of the insulin-like growth factor-binding proteins by follicle size in the human ovary
Authors: Hughes S C Cwyfan , Mason H D , Franks S , Holly J M P
Published: December 2004

The IGFs are believed to play an important role in the regulation of steroidogenesis and follicular maturation in the human ovary. The activities of the IGFs are regulated by a family of binding proteins (IGFBPs) which are subject to a number of potential post-translational modifications. The aim of this study was to investigate both the production and modification of the IGFBPs in follicular fluid and in medium conditioned by granulosa cells and theca from individual follicles at different stages of maturation.

In follicular fluid from healthy, dominant follicles there was an increase in the amount of IGFBP-2, -3 and -4 present as lower molecular weight forms (23 kDa, 29 kDa and 16·5 kDa respectively) in comparison to that seen in atretic follicles from the same ovary. Furthermore for IGFBP-4, this fragmentation was confirmed to be attributable to the presence of a specific protease which could be inhibited not only by the addition of metal ion chelators or serine protease inhibitors, but also by the addition of other recombinant unsaturated IGFBPs, particularly IGFBP-3. IGF-I did not modulate the activity of the IGFBP-4 protease in solution but was able to prevent the inhibition seen with IGFBP-3.

Analysis of granulosa cell conditioned medium from the same series of healthy and atretic follicles revealed that IGFBP-2 and -4 were the predominant IGFBPs with no fragments seen on immunoblotting. In contrast, IGFBP-3 in conditioned medium from theca of atretic follicles was always found as an intact doublet, but was found partially fragmented (30 and 32 kDa) in medium conditioned by theca from healthy, dominant follicles with the proportion of IGFBP-3 in this lower molecular weight or fragmented doublet increasing with follicular maturation. A similar situation was also found for IGFBP-4 with the progressive increase in the amount of the 15 and 16·5 kDa fragments. IGFBP-2 was always found to be intact. Finally, IGFBP production from stroma explants was also examined. This revealed a wide variation in IGFBP pattern between different ovaries, although there was a remarkable degree of consistency between different stroma explant cultures from the same ovary. Immunoblotting for IGFBP-3 revealed that, where present, it existed as both an intact and a lower molecular weight doublet and that IGFBP-2 was again always found to be intact.

In conclusion we have demonstrated alterations in the proteolytic modification of the IGFBPs which differ in the various follicular compartments and are closely linked to the stage of follicular development.

Journal of Endocrinology (1997) 154, 35–43
Effects of vasopressin and elimination of corticotropin-releasing hormone-target cells on pro-opiomelanocortin mRNA levels and adrenocorticotropin secretion in ovine anterior pituitary cells
Authors: van de Pavert S A , Clarke I J , Rao A , Vrana K E , Schwartz J
Published: December 2004

Although arginine-vasopressin (AVP) is reported to produce greater ACTH biosynthetic and secretory responses than does corticotropin-releasing hormone (CRH) in sheep anterior pituitary cells, neither factor appears to increase pro-opiomelanocortin (POMC) mRNA levels, as does CRH in the cells of some other species. Since only a fraction of cells that express POMC mRNA may be able to respond to AVP, the aim of this study was to further delineate the regulation of POMC mRNA in ovine anterior pituitary corticotrophs, as a whole and in functional subpopulations of corticotrophs. We measured the effects of AVP, CRH or activation of protein kinase C by phorbol myristate acetate (PMA) in cultured cells. We compared responses in intact populations with those of cultures from which CRH-target cells were pharmacologically eliminated. Dissociated adult ovine anterior pituitary cells were cultured overnight, treated with either vehicle (intact) or a CRH-toxin conjugate that specifically eliminates CRH-target cells (CRH-target-depleted), washed, returned to culture and subsequently challenged with vehicle, AVP (100 nm), CRH (10 nm) or PMA (1 μm) for 5 h. The media were assayed for ACTH by RIA and the cells for POMC mRNA by Northern blot analysis. In intact populations, AVP and CRH increased ACTH secretion from 6·5 ±1·2 to 216 ±22 and 81 ± 14 ng/well respectively, but only AVP caused an increase in steady-state POMC mRNA levels (+48 ± 10%). Direct activation of protein kinase C with PMA mimicked the effect of AVP on ACTH secretion (318 ± 16 ng/well), but did not alter POMC mRNA levels. In CRH-target-depleted populations, control ACTH secretion (11 ± 3 ng/well) and POMC mRNA (+69 ±7%) were elevated, compared with intact populations. AVP (55 ± 8 ng/well) and PMA (120 ± 17 ng/well), but not CRH, increased ACTH secretion; POMC mRNA was not significantly elevated by any of the treatments. Taken together, these data provide further support for the notion of dissociation between secretion of ACTH and expression of POMC mRNA, and demonstrate that AVP increases steady-state POMC mRNA levels in ovine anterior pituitary cells. The data are also consistent with the concept that complex interactions, possibly including those between cells, influence ACTH secretion and steady-state POMC mRNA levels.

Journal of Endocrinology (1997) 154, 139–147
Tri-iodothyronine increases insulin-like growth factor binding protein-4 expression in rat hepatocytes
Authors: Demori I , Bottazzi C , Voci A , Gallo G , Scharf J-G , Fugassa E
Published: December 2004

Previous in vivo studies demonstrated significant variations in insulin-like growth factor binding protein-1 (IGFBP-1), IGFBP-2 and IGFBP-4 hepatic mRNAs and/or serum levels depending on the rat thyroid status. In this study we employed cultured hepatocytes from adult rats to demonstrate a possible direct regulation of these genes by tri-iodothyronine (T3). Northern blot analysis revealed that IGFBP-1 and -4 messages were clearly expressed, whereas IGFBP-2 signal was barely detectable.

No significant effects on IGFBP-1 mRNA level or on peptide secretion were detected in T3-cultured hepatocytes. In contrast, significant increases in IGFBP-4 mRNA steady-state levels as well as in IGFBP-4 secretion were observed in hepatocytes cultured for 12–24 h in the presence of T3. The T3 effect on IGFBP-4 transcript levels appears to consist of enhanced gene transcription and is independent of ongoing protein synthesis.

The T3-increased IGFBP-4 expression in cultured hepatocytes is consistent with our in vivo experiments demonstrating an increase in hepatic IGFBP-4 mRNA and serum IGFBP-4 levels in T3-treated rats. Furthermore, significant decreases in hepatic IGFBP-4 message and serum IGFBP-4 levels were observed in hypothyroid rats compared with euthyroid controls.

Our data establish an important direct role for thyroid hormone in regulating IGFBP-4 expression and consequently IGF activity.

Journal of Endocrinology (1997) 154, 155–165
Thyroid hormone induces protein secretion and morphological changes in astroglial cells with an increase in expression of glial fibrillary acidic protein
Authors: Lima F R S , Trentin A G , Rosenthal D , Chagas C , Neto V Moura
Published: December 2004

Thyroid hormone (T3) induces in vitro differentiation of astrocytes from the developing rat brain. T3 treatment induced the appearance of long processes in cultured cerebral hemisphere and mesencephalon astrocytes from embryonic and newborn rats. T3 treatment also produced a change in the morphology of cultured cerebellar astrocytes from 10-day-old rats, but not in cerebellar astrocytes from newborn rats. An increased expression of glial fibrillary acidic protein (GFAP) was also seen in the T3-treated newborn cerebral hemisphere and mesencephalic astrocytes. The morphological changes were induced earlier when the astrocytes were treated with conditioned medium (CM) obtained from cultures previously exposed to T3. Our results show that astrocytes from the developing rat brain are not homogenous in their responsiveness to T3. Furthermore, the fact that CM produces a response similar to that obtained with T3 treatment but in less time, suggests that T3 might induce the secretion of factors by cultured astrocytes. These factors might, by an autocrine/paracrine effect, induce the expression of GFAP and differentiation in developing brain astrocytes.

Journal of Endocrinology (1997) 154, 167–175
The effect of maternal protein deficiency during pregnancy and lactation on glucose tolerance and pancreatic islet function in adult rat offspring
Authors: Wilson M R , Hughes S J
Published: December 2004

To test the hypothesis that poor foetal–neonatal nutrition predisposes adult animals to impaired glucose tolerance or diabetes, pregnant and lactating rats were fed a low (5%) protein diet and glucose tolerance and pancreatic islet function then assessed in the adult offspring. To expose any underlying defects the offspring were allowed access to a sucrose supplement (35%) or fed a high fat diet. Offspring born to low protein-fed females had significantly lower body weights than controls. In islets from previously malnourished rats, insulin release in batch incubations or perifusion was not significantly different to controls. In islets from previously malnourished animals fed sucrose, glucose-stimulated insulin release was reduced in perifusion by 66% (P<0·01) and batch incubations by 26–52% (6–16 mmol/l glucose, (P<0·01). Similarly, impaired secretory responses were found in islets from previously malnourished animals fed a high fat diet. These did not result from a reduced pool of releasable insulin, as arginine-stimulated secretion was not impaired. Rats previously malnourished showed a normal glucose tolerance. Glucose tolerance was impaired, however, in previously malnourished rats fed sucrose (area under the glucose tolerance test curve was increased by 42%, P<0·05) but despite the reduced islet secretory responses was not significantly different to sucrose-fed controls (area increased by 54%, P<0·05). Glucose tolerance was impaired in previously malnourished animals fed high fat diet (area increased by 48%, P<0·05) more so than in high fat fed-controls (28% increase, NS). These data support the hypothesis that poor foetal–neonatal nutrition leads to impaired pancreatic β-cell function which persists into adult life. Alone this is not sufficient to produce diabetes, but an inability to respond to a highly palatable fat diet may tip the balance towards impaired glucose tolerance.

Journal of Endocrinology (1997) 154, 177–185
Arachidonic acid release from rat Leydig cells depends on the presence of luteinizing hormone/human chorionic gonadotrophin receptors
Authors: Moraga P F , Llanos M N , Ronco A M
Published: December 2004

In this work, the involvement of arachidonic acid (AA) in the luteinizing hormone and human chorionic gonadotrophin (LH/hCG) action on Leydig cells was studied. Experiments were first designed to evaluate [14C]AA incorporation into membrane phospholipids. Subsequently, time-course, pulse-chase and dose–response studies of the effect of hCG on [14C]AA release were performed. Results indicated that 4 h was optimal for maximal incorporation of [14C]AA into membrane phospholipids of viable Leydig cells. Pulse-chase experiments and studies performed to evaluate the effect of different doses of hCG on [14C]AA release demonstrated that this hormone stimulates [14C]AA release in a dose–response and time-dependent manner. Furthermore, using a desensitised animal model, a link between the presence of LH/hCG receptors and LH/hCG-stimulated [14C]AA release in Leydig cells could be established. In fact, the amount of [14C]AA released was significantly dependent on, and directly proportional to, the concentration of LH/hCG binding sites. Thus [14C]AA released from intact rat Leydig cells decreased when animals had been previously injected with a high single dose of hCG (desensitised animals), which is known to cause a dramatic decrease in the number of LH/hCG binding sites. These results demonstrate that the mechanism of AA release in Leydig cells depends on LH/hCG–receptor interaction and also suggest that AA could act as an additional intracellular messenger associated with the hormonal action of LH/hCG.

Journal of Endocrinology (1997) 154, 201–209
Increase by tri-iodothyronine of endothelin-1, fibronectin and von Willebrand factor in cultured endothelial cells
Authors: Baumgartner-Parzer S M , Wagner L , Reining G , Sexl V , Nowotny P , Müller M , Brunner M , Waldhäusl W
Published: December 2004

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T3) for 13 ± 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T3 significantly increased vWF secretion (50 ng T3/ml: 117 ± 5%, P<0·01; 100 ng T3/ml: 127 ± 26%, P<0·01) as well as vWF mRNA expression (50 ng/ml: 116 ± 13%, P<0·001; 100 ng/ml: 136 ± 30%, P<0·002) (results are means ± s.d. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 ± 42% of control, P<0·05) and 100 (116·8 ± 16% of control, P<0·05) ng T3/ml, and FN mRNA expression by 50 ng T3/ml (123 ± 20%, P<0·05). Long-term incubation with T3 increased both ET-1 secretion (25 ng/ml: 124 ± 25%, P<0·001; 50 ng/ml: 165 ± 53%, P<0·05; 100 ng/ml: 116 ± 17%, P<0·05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 ± 16%, P<0·05; 50 ng/ml: 134 ± 43%, P<0·02; 100 ng/ml: 120 ± 20%, P<0·02). Protein kinase C (PKC) isoforms ε and βII were not significantly affected by T3, whereas PKC α was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T3/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [3H]thymidine assays, remained unaffected in HUVECs after exposure to T3. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC α-, βII- and ε-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.

Journal of Endocrinology (1997) 154, 231–239
Estrogen increases in vivo leptin production in rats and human subjects
Authors: Shimizu H , Shimomura Y , Nakanishi Y , Futawatari T , Ohtani K , Sato N , Mori M
Published: December 2004

The decrease in estrogen in menopausal women increases body fat. The present studies were undertaken to investigate the involvement of estrogen in leptin production in vivo. In the first study, expression of ob gene mRNA in white adipose tissue was measured at 2 and 8 weeks after ovariectomy in rats. In the second, serum leptin concentration was measured in total body fat of 87 weight-matched human subjects (29 men, 29 premenopausal and 29 postmenopausal women). In the third, changes in serum leptin concentration with the menstrual cycle were determined, ob gene expression decreased in subcutaneous and retroperitoneal white adipose tissue of ovariectomized rats 8 weeks after the operation, while ovariectomy increased ob gene expression in mesenteric white adipose tissue. Serum leptin concentration was decreased by ovariectomy. Estradiol supplement reversed the effect of ovariectomy on ob gene expression and circulating leptin levels. In humans, serum leptin concentration was higher in premenopausal women than in men, and in postmenopausal women it was lower than in premenopausal women, but still higher than in men. In 13 premenopausal women, serum leptin levels were significantly higher in the luteal phase than in the follicular phase. The present studies strongly indicate that estrogen regulates leptin production in rats and human subjects in vivo. Regional variation in the regulation of ob gene expression by estrogen was found.

Journal of Endocrinology (1997) 154, 285–292
Effect of chronic ethanol on reproductive and growth hormones in the peripubertal male rat
Authors: Steiner J C , LaPaglia N , Hansen M , Emanuele N V , Emanuele M A
Published: December 2004

Ethanol (EtOH) has previously been shown to have profound effects on various endocrine systems. The present study further investigates the action of EtOH on testosterone and on the GH–IGF-I axis. Since these hormones are particularly important in male rats progressing through puberty, we examined the effect of 10 days of EtOH treatment at three different ages (35, 50 and 65 days old) as male rats progressed through puberty into adulthood. After 10 days of feeding a 6% EtOH liquid diet, serum testosterone levels were markedly decreased in all three ages (P<0·02 at 35 days, P<0·01 at 50 days and P<0·03 at 65 days). IGF-I was assessed and was differentially affected at each age. At 35 days IGF-I levels were suppressed by EtOH (P<0·0002), at 50 days no change was apparent, and at 65 days levels were significantly higher in EtOH-treated (P<0·01) compared with liquid-fed controls. The levels of IGF-I in the EtOH-treated animals paralleled pituitary GH mRNA levels with a significant fall in the expression of GH mRNA levels noted at 35 days (P<0·04), no change at 50 days and a significant rise observed at 65 days (P<0·03). At the hypothalamic level, GH-releasing hormone (GRF) mRNA was significantly reduced in the two younger EtOH-treated age groups compared with controls (P<0·04 at 35 days; P<0·02 at 50 days). At 65 days of age, EtOH did not alter GRF mRNA levels. No EtOH-induced changes were seen in GRF content at any age. These observations indicate definite age-related alterations in hormonal gene expression and circulating serum hormone levels and emphasize the importance of studying these critical peripubertal ages after chronic EtOH exposure.

Journal of Endocrinology (1997) 154, 363–370
Glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis
Authors: Dempster DW , Moonga BS , Stein LS , Horbert WR , Antakly T
Published: December 2004

We have studied the effects of glucocorticoids on the activity and viability of neonatal rat osteoclasts in vitro. In the bone slice assay, glucocorticoids caused a dose-dependent decrease in the amount of bone resorbed, which was accompanied by a parallel decrease in osteoclast number. Loss of osteoclasts was due to their death, which occurred by the process of apoptosis. Evidence for the latter was obtained by a range of techniques, including time-lapse video microscopy, acridine orange staining, DNA fragment detection and transmission electron microscopy. Immunocytochemistry revealed the presence of glucocorticoid receptors in osteoclasts, and glucocorticoid-induced cell death could be prevented by the glucocorticoid receptor antagonist, RU486. These observations suggest that glucocorticoids promote receptor-mediated apoptosis of rat osteoclasts in vitro. This finding may help to explain recent data indicating that, in sharp contrast with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo.
Characterisation of natriuretic peptide receptors in eel gill
Authors: Mishina S , Takei Y
Published: December 2004

A radioligand-binding assay was established for eel atrial natriuretic peptide (eANP), and ANP receptors were characterised in the eel gill. ANP binding to the gill membrane fraction was saturable with increasing ligand concentrations and was specific to ANP peptides, i.e. eANP, ventricular natriuretic peptide (VNP) and C-type natriuretic peptide (CNP). A Scatchard analysis revealed a single class of high-affinity receptors with a Kd of 59.2 pM and a Bmax of 67.9 fmol/mg protein. The Kd value is within the range of plasma ANP concentration of the eel. Kd and Bmax did not differ between freshwater (FW)- and seawater (SW)-adapted fish. The gill receptors exhibited similar affinity for eANP, eVNP and eCNP, and ANP binding was almost completely displaced by C-ANF, a specific ligand for guanylate cyclase-uncoupled receptors. The presence of this type of receptor (natriuretic peptide receptor (NPR)-C and NPR-D) was also indicated in eel gill by affinity labelling. cGMP production was stimulated by the addition of eCNP but not by eANP and eVNP, and this was observed only in FW eels. Thus, most ANP receptors in eel gill are NPR-C and NPR-D, but a small number of the NPR-B type are also present in FW eels. In addition to the gill, specific ANP receptors were detected in the red body of the swim bladder, the brain, digestive tracts, kidney, head kidney and urinary bladder, in most of which ANP action has been reported. The density of ANP receptors decreased in most tissues after adaptation to SW except the gill, brain, atrium and spleen.
Dissociation of nitric oxide synthase immunoreactivity and NADPH-diaphorase enzyme activity in rat pituitary
Authors: Wang H , Christian HC , Morris JF
Published: December 2004

The relationship between nitric oxide synthase (NOS) immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry was investigated in the anterior and posterior pituitary of ovariectomized rats. NADPH-d activity was present throughout the posterior pituitary but could not be detected in the anterior pituitary. By contrast, an antibody raised against whole recombinant rat neuronal NOS revealed strongly NOS-immunoreactive gonadotrophs and folliculo-stellate cells scattered throughout the anterior pituitary in addition to immunoreactive fibres in the posterior pituitary. The study demonstrates that NADPH-d histochemistry is not always coincident with NOS and provides evidence for an as yet uncharacterized subtype of NOS in the rat anterior pituitary.
Extracellular calmodulin-binding proteins in body fluids of animals
Authors: WenQiang T , Yi G , Yu S , Jung T , DaYe S
Published: December 2004

The extracellular calmodulin-binding proteins (CaMBPs) were investigated in body fluids of animals by using the biotinylated calmodulin gel overlay method. Four major CaMBPs with molecular masses of 24, 31.5, 44/45 and 94 kDa were detected in serum, two of 24 and 63 kDa in bovine milk and three of 14, 24 and 52 kDa in human saliva. It suggested that extracellular CaMBPs exist commonly in body fluids of animals, and this result may provide a new clue for understanding the role of extracellular calmodulin.