bluuebunny/crossref_metadata_2025 huggingface dataset:
Luteotrophic factors in hyperstimulated pseudopregnant rabbit: I – Evidence for aromatase activity in luteal tissue and luteal cells
Authors: Arioua R K , Féral C , Benhaïm A , Delarue B , Leymarie P
Published: December 2004

It is well established that the rabbit corpus luteum (CL) function depends upon endogenous oestradiol, the major source of which in the rabbit ovary is considered to be the ovarian follicles. The absence of oestradiol formation by the rabbit CL has been previously reported.

In a hyperstimulated pseudopregnant rabbit model used in our laboratory which developed a large number of corpora lutea in response to chorionic gonadotrophin (eCG)/hCG, we observed the survival of corpora lutea in vivo, and normal levels of plasma progesterone throughout pseudopregnancy (PP), despite the scarcity or the absence of follicles as a source of the luteotrophic hormone. Measurement of oestradiol in the plasma indicated that it was at high levels and correlated with the number of corpora lutea. This led us to investigate the luteal origin of oestradiol in this model.

PP was induced in rabbits by i.m. injection of 200 IU eCG daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue obtained at days 5, 9 and 12 of PP and cultured for 24 h synthesized oestradiol and testosterone in addition to progesterone. However, under the same conditions, follicles had limited capacity to secrete oestradiol. The presence of an aromatase activity in luteal tissue was confirmed when exogenous testosterone was added to the culture medium. P450aromatase (P450arom) mRNA was found in luteal tissue at days 5, 9 and 12 of PP.

Small or large luteal cells, obtained by enzymatic digestion of the tissue followed by centrifugation in a Percoll density gradient, were cultured during several days with or without gonadotrophin or dibutyryl cAMP (dbcAMP). Both types of cells secreted oestradiol. In small cells and luteal tissue, aromatase activity was stimulated (1·5–2-fold) by hCG and dbcAMP. Large cells exhibited a greater capacity to aromatize testosterone than small cells, but aromatase activity was not modified by hCG or by dbcAMP. FSH had no effect on aromatase activity of either luteal cell type.

This intrinsic luteal tissue aromatase capacity and the absence of premature regression of corpora lutea despite the limited support of follicular oestrogen, suggest an autocrine and luteotrophic role for this luteal oestrogen.

Journal of Endocrinology (1997) 154, 249–257
Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies
Authors: Mañes S , Kremer L , Vangbo B , López A , Gómez-Mouton C , Peiró E , Albar J P , Mendel-Hartvig I B , Llopis R , Martínez-A C
Published: December 2004

The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I–VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first α-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55–70) and (1–13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0·5–10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed.

Journal of Endocrinology (1997) 154, 293–302
Autoregulation of central and peripheral growth hormone receptor mRNA in domestic fowl
Authors: Hull KL , Harvey S
Published: December 2004

Growth hormone (GH) regulates numerous cellular functions in many different tissues. A common receptor is believed to mediate these tissue-specific effects, suggesting that post-receptor signalling molecules or tissue sensitivity to GH may differ between tissues. Tissue sensitivity depends upon the abundance of GH receptors (GHRs), thus tissue-specific GHR regulation could enable tissue-specific GH actions. The comparative autoregulation of GHR gene transcription in central (whole brain or hypothalami) and peripheral (liver, bursa, spleen and thymus) tissues was therefore examined in domestic fowl. In all tissues, a 4.4 kb GHR gene transcript that encodes the full-length GHR was identified. The abundance of this transcript was inversely related to endogenous GH status; it was lower in males with high circulating concentrations of GH and higher in females with lower basal concentrations of plasma GH. The abundance of this transcript was also rapidly downregulated in response to a bolus systemic injection of recombinant chicken GH, designed to mimic an episodic burst of endogenous GH release. This autoregulatory response was observed within 2 h of GH administration and was of greater magnitude in the brain than in peripheral tissues. Intracerebroventricular injections of GH also downregulated GHR gene expression in the brain, although hepatic GHR transcripts were unaffected 24 h after central administration of GH. In contrast, the induction of hyposomatotropism by passive GH immunoneutralization increased the abundance of the GHR transcript in the thymus, but not in other central (brain) or peripheral (bursa, liver) tissues. GH is not the sole regulator of GHR abundance, however; hypersomatropism induced by hypothyroidism was associated with an increase in GHR mRNA. The expression of the GHR gene in the domestic fowl would thus appear to be autoregulated by GH in a tissue-specific way.
Culture of human insulinoma cells: development of a neuroendocrine tumor cell- and human pancreatic islet cell-specific monoclonal antibody
Authors: Wagner L , Templ E , Reining G , Base W , Weissel M , Nowotny P , Kaserer K , Waldhausl W
Published: December 2004

We report on the culture of human insulinoma cells derived from a 32-year-old male patient with hyperinsulinism due to an insulinoma of the pancreas. A single-cell suspension was made by passing insulinoma fragments through a fine-gauge stainless-steel mesh. Cluster-forming insulinoma cells resembling pancreatic islets grew in the presence of fibroblasts. The insulinoma cell clusters could be differentiated from fibroblasts by using in situ pan optic staining and specific immunocytochemical staining (anti-human insulin and anti-human insulinoma monoclonal antibody (mAb) D24). mAb D24 was generated using insulinoma cells as antigen for immunization of a Balb/C mouse and cell fusion by the hybridoma cell technique. The anti-insulinoma cell mAb recognized a 32 kDa protein on immunoblot analysis of neuroendocrine tumor cells. D24 mAb also reacted immunohistochemically with normal pancreatic beta-cells and tumors such as vipoma, gastrinoma and carcinoid. Insulinoma cell clusters separated from fibroblasts by micromanipulation and plated into multiwell culture dishes exhibited an insulin-secretion rate of approximately 30 U/100 cells per 24 h with no insulin-secretory response to elevated glucose concentration. Purified insulinoma cells incubated with 1 ng/ml human nerve growth factor expressed neurofilament and neurite extension. These findings together with earlier observations in animal models suggest that human pancreatic beta-cells share some properties with neurons and are related to other neuroendocrine cells in the gastrointestinal tract.
Failure of exogenous IGF-I to restore normal growth in rats submitted to dietary zinc deprivation
Authors: Ninh NX , Maiter D , Verniers J , Lause P , Ketelslegers JM , Thissen JP
Published: December 2004

Dietary zinc deficiency in rats causes growth retardation associated with decreased circulating IGF-I concentrations. To investigate the potential role of low IGF-I in this condition, we attempted to reverse the growth failure by administration of exogenous IGF-I. Rats were fed for 4 weeks a zinc-deficient diet (ZD, Zn 0 ppm) or were pair-fed a zinc-normal diet (PF, Zn 75 ppm). We compared the anabolic action of recombinant human (rh) IGF-I infused at the dose of 120 microg/day for the last experimental week in ZD, PF and freely fed control (CTRL) rats. Zinc deficiency caused growth stunting (weight gain 47% of PF; P<0.001), decreased circulating IGF-I (52% of PF; P<0.01) and liver IGF-I mRNA (67% of PF; P<0.01). Serum insulin-like growth factor-binding protein-3 (IGFBP-3) assessed by ligand blot was also reduced in ZD rats (65% of PF; P<0. 01). While exogenous IGF-I increased body weight in CTRL (+12 g; P<0. 01) and PF (+7 g; not significant) animals, growth was not stimulated in ZD rats (-1.5 g) in comparison with the corresponding untreated groups. However, circulating IGF-I and IGFBP-3 levels were restored by IGF-I infusion to levels similar to those in untreated CTRL rats. In conclusion, restoration of normal circulating levels of IGF-I and IGFBP-3 by rhIGF-I infusion fails to reverse the growth retardation induced by zinc deficiency. These results suggest that growth retardation related to zinc deficiency is not only caused by low serum IGF-I concentrations, but also by inhibition of the anabolic actions of IGF-I.
Retinoic acid stimulates IGF binding protein (IGFBP)-6 and depresses IGFBP-2 and IGFBP-4 in SK-N-SH human neuroblastoma cells
Authors: Chambery D , de Galle B , Babajko S
Published: December 2004

Insulin-like growth factors (IGF-I and IGF-II) stimulate proliferation and differentiation in many cell types. In biological fluids, they associate non-covalently with high-affinity binding proteins (IGFBPs) which control their bioavailability and modulate their action. We previously demonstrated that IGFBP-2, -4 and -6 are intimately involved in the growth of cells derived from human neuroblastomas. Here, we have investigated the effects of retinoic acid (RA), which induces differentiation in these cells, on the expression of IGFBPs secreted by SK-N-SH neuroblastoma cells. Analysis of transcriptional activity of the IGFBP-2, -4 and -6 genes in isolated nuclei (run-on experiments) showed that RA increased the transcriptional activity of the IGFBP-6 gene, reduced that of the IGFBP-4 gene and had no effect on that of the IGFBP-2 gene. Northern blot analysis following treatment with actinomycin D showed that RA increased the stability of IGFBP-6 mRNA by a factor of 2.6, decreased that of IGFBP-2 mRNA by a factor of 2.3 and failed to affect IGFBP-4 mRNA. Treatment of cells with cycloheximide indicated the involvement of labile proteins in the stabilization of these mRNAs the expression of which could be under the control of RA. The transcriptional and/or post-transcriptional mechanisms by which RA regulates each of the IGFBPs produced by SK-N-SH cells are therefore different. Such regulation may also reflect the state of differentiation of the neuroblastoma cells. With RA-induced differentiation, IGFBP-6 is strongly stimulated, whereas IGFBP-2 and IGFBP-4 are severely depressed, which would suggest that each IGFBP plays a specific role. Moreover, this regulation seems tissue-specific because it is different in other cell types.
Isolated porcine ovarian follicles as a model for the study of hormone and growth factor action on ovarian secretory activity
Authors: Sirotkin AV , Makarevich AV , Kotwica J , Marnet PG , Kwon HB , Hetenyi L
Published: December 2004

The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.
Insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein-2 and pregnancy-associated glycoprotein mRNA in pigs with somatotropin-enhanced fetal growth
Authors: Sterle JA , Boyd C , Peacock JT , Koenigsfeld AT , Lamberson WR , Gerrard DE , Lucy MC
Published: December 2004

Fetal growth is increased when pregnant gilts are treated with recombinant porcine somatotropin. The mechanism for increased fetal growth was examined by measuring the expression of IGF-I and -II and IGF-binding protein-2 (IGFBP-2) mRNA in liver and reproductive tissues of somatotropin- and saline-treated pregnant gilts. Twenty-four pregnant gilts received daily injections of either saline (control; n=12) or 5 mg recombinant porcine somatotropin (n=12) from day 30 to day 43 of gestation. Gilts were slaughtered on day 44 of gestation and liver, ovary, placenta, placental uterus (uterus with adjacent placental tissue) and non-placental uterus (region of the necrotic tip) were collected. The mRNAs for somatotropin receptor, IGFs -I and -II, IGFBP-2 and pregnancy-associated glycoprotein (a marker of trophoblast tissue) were analyzed by Northern blotting or ribonuclease protection assay. Gilts treated with somatotropin had heavier fetuses and placentas. The concentration of mRNA for the components of the IGF system was tissue-dependent. The uterine IGF-I mRNA concentration was greater in non-placental than in placental uterus. The greatest IGF-II mRNA concentration was observed in placenta, and adjacent uterine tissue expressed IGFBP-2 mRNA intensely. In non-placental uterus, IGFBP-2 mRNA was nearly undetectable. Somatotropin-dependent regulation of IGF-I was only observed in liver, where the greatest somatotropin receptor mRNA concentration was found. In the pregnant uterus, somatotropin failed to change the concentration of IGF or IGFBP-2 mRNA. Pregnancy-associated glycoprotein mRNA concentration was decreased by somatotropin. In summary, increased fetal growth in somatotropin-treated pregnant pigs was not associated with changes in IGF or IGFBP-2 mRNA concentration in reproductive tissues. Other mechanisms, therefore, lead to enhanced fetal growth in somatotropin-treated pregnant pigs.
Regulation of prostaglandin production in intact fetal membranes by interleukin-1 and its receptor antagonist
Authors: Brown NL , Alvi SA , Elder MG , Bennett PR , Sullivan MH
Published: December 2004

There is strong evidence for the involvement of inflammatory mediators such as interleukin (IL)-1 in the biochemical mechanisms of parturition. Therefore the effects of the IL-1 family (IL-1alpha (1 ng/ml), IL-1beta (1 ng/ml) and the IL-1 receptor antagonist (IL-1ra) (10 ng/ml)) on the regulation of prostaglandin synthesis in term human fetal membranes were investigated. It was found that, after 4 h of culture, IL-1beta increased prostaglandin E2 (PGE2) output approximately twofold. This was associated with both a significant increase in cyclo-oxygenase-2 (COX-2) mRNA levels (approximately fourfold compared with control) and translocation of cytoplasmic phospholipase A2 (cPLA2) from the cytosol to the membrane fraction. IL-1alpha was less effective than IL-1beta at stimulating PGE2 production through similar mechanisms. IL-1ra had no effect on PGE2 output. However, in combination treatments, IL-1ra did not inhibit IL-1alpha- or IL-1beta-stimulated PGE2 output, and increased PGE2 production further compared with IL-1beta alone. IL-1ra decreased IL-1beta-induced COX-2 mRNA expression by about half and significantly increased cPLA2 protein levels, as detected by immunoblotting, when used alone and together with IL-1beta. These results suggest that IL-1ra has partial agonist properties when used together with IL-1alpha and IL-1beta in fetal membranes by increasing cPLA2 protein levels, which leads to an increase in the production of prostaglandins.
The influence of the colon on postprandial glucagon-like peptide 1 (7-36) amide concentration in man
Authors: Robertson MD , Livesey G , Morgan LM , Hampton SM , Mathers JC
Published: December 2004

Glucagon-like peptide (7-36) amide (GLP-1) is an incretin hormone of the enteroinsular axis released rapidly after meals despite the fact that GLP-1 secreting cells (L-cells) occur predominantly in the distal gut. The importance of these colonic L-cells for postprandial GLP-1 was determined in healthy control subjects and in ileostomy patients with minimal small bowel resection (<5 cm). Subjects were fed a high complex carbohydrate test meal (15.3 g starch) followed by two carbohydrate-free, high fat test meals (25 g and 48.7 g fat respectively). Circulating levels of glucose, insulin, glucagon, glucose insulinotrophic peptide (GIP) and GLP-1 were measured over a 9-h postprandial period. For both subject groups the complex carbohydrate test meal failed to elicit a rise in either GIP or GLP-1. However, both hormones were elevated after the fat load although the GLP-1 concentration was significantly reduced in the ileostomist group when compared with controls (P=0.02). Associated with this reduction in circulating GLP-1 was an elevation in glucagon concentration (P=0.012) and a secondary rise in the plasma glucose concentration (P=0.006). These results suggest that the loss of colonic endocrine tissue is an important determinant in the postprandial GLP-1 concentration. Ileostomists should not be assumed to have normal enteroinsular function as the colon appears to have an important role in postprandial metabolism.
Non-steroidal factors in bovine follicular fluid inhibit or facilitate the action of pulsatile administration of GnRH on LH release in the female rat
Authors: van Dieten JA , Helder MN , van den Oever C , de Koning J
Published: December 2004

Using steroid-free bovine follicular fluid (bFF), we studied the action of gonadotrophin surge-inhibiting factor/attenuating factor (GnSIF/AF) on GnRH-induced self-priming in phenobarbital-blocked female rats. For the experiments we used intact rats, short-term (4 h) ovariectomized (OVX) rats and long-term (14 days) OVX rats. In the latter case the rats were injected with 17beta-oestradiol benzoate (OB, 40 micrograms) or vehicle only, 2 or 48 h before the experiment. GnRH (10-50 pmol/kg body weight) was injected intra-arterially in 5 or 15 pulses, respectively 60 or 20 min apart, starting 1 or 4 h after injection of bFF (0.5 or 1.0 ml). In response to 25 pmol/kg GnRH pulses (1/h), we observed no effect in the long-term OVX rats, a minor effect in the intact rats and an enhanced self-priming effect in the short-term OVX rats. Administration of bFF attenuated or even completely inhibited the self-priming process. However, in the case of long-term OVX rats LH release was inhibited only after long-term OB priming. Furthermore, 4 h after administration of bFF, LH release in response to 25 pmol/kg GnRH pulses (3/h) was inhibited transiently in intact rats and long-term OVX rats. The results support the hypothesis of a functional antagonistic action between GnRH and GnSIF/AF. However, when injected 1 h before, bFF facilitated the initial release of the surge-like LH pattern in intact rats in response to 3 pulses/h of GnRH. These results are consistent with an important role of GnSIF/AF and other non-steroidal ovarian factors in the control of both low LH concentrations and the generation of the LH surge. Some genomic action of oestradiol might be a prerequisite for the inhibitory effect of GnSIF/AF on GnRH-induced LH release.
Using skeletal muscle as an artificial endocrine tissue
Authors: MacColl GS , Goldspink G , Bouloux PM
Published: December 2004

Gene transfer into muscle tissue is currently being developed as a method for the production, secretion and delivery of therapeutic proteins. This methodology has been used to produce a variety of physiologically active proteins and may ultimately be applied to the treatment of several diseases. In this review, we consider several applications of this methodology and discuss approaches for modulating therapeutic protein production and secretion from muscle, using growth hormone as an example. In addition, factors limiting the effectiveness of muscle gene transfer are also discussed, as these shall determine the efficacy of muscle gene transfer when applied to humans.
Characterization and hormonal modulation of immunoreactive thiamin carrier protein secreted by adult rat Leydig cells in vitro
Authors: Subramanian S , Adiga PR
Published: December 2004

Leydig cells isolated from adult rats and maintained under defined conditions in culture secrete a protein of molecular weight (Mr) 70 000 which is immunologically similar to chicken thiamin carrier protein (TCP). Synthesis of immunoreactive TCP by these cells is demonstrated by immunoprecipitation of [35S]methionine incorporated, newly synthesized proteins with monoclonal and polyclonal antibodies to chicken TCP. The amount of immunoreactive TCP secreted into the culture supernatant is quantitated by using a specific radioimmunoassay. Under the influence of LH, secretion of immunoreactive TCP is enhanced 3-fold and can be inhibited by up to 70% with aromatase inhibitor (1,4,6-androstatrien-3,17-dione). Cyclic AMP acts as a second messenger in the sequence of events involved in LH-induced elevation of immunoreactive TCP in Leydig cells. The effects of exogenous estradiol-17beta and diethylstilbestrol are comparable in terms of stimulation of secretion of immunoreactive TCP by these cells. Tamoxifen brought about a 70% decrease in the elevated levels of immunoreactive TCP. These results suggest that estrogen mediates immunoreactive TCP induction in hormonally stimulated adult rat Leydig cells.
The different approaches to the genetic analysis of autoimmune thyroid disease
Authors: Allahabadia A , Gough SC
Published: December 2004

Graves' disease and Hashimoto's thyroiditis are organ-specific autoimmune disorders of multifactorial aetiology with a polygenic mode of inheritance. Familial clustering and twin studies provide evidence for a genetic predisposition. Three main approaches have been used in the search for susceptibility loci: population-based case-control studies, classical linkage analysis, and intrafamilial linkage disequilibrium. Case-control studies are a sensitive method of gene detection and the collection of subjects is resource-efficient. However, they require prior knowledge of a candidate gene and are prone to inconsistent results due to false positives that may arise from population mismatch. Linkage analysis is a powerful tool for detecting 'major' genes that does not require a candidate gene and is, therefore, a means of genome screening. This method, however, has limited power to detect genes of 'modest' effect, and the collection of sibpairs and multiple family members may be difficult. Intrafamilial linkage disequilibrium analysis is more sensitive than classical linkage analysis, requires only one affected offspring, and eliminates population mismatch. This approach has confirmed linkage disequilibrium of the HLA region with Graves' disease, previously not detected by linkage analysis. Knowledge of a candidate locus is required, however, and this method cannot, therefore, at present be used for genome screening. It is likely that a combination of all three approaches will be required to identify susceptibility loci for autoimmune thyroid disease.
Viral vectors for gene delivery and gene therapy within the endocrine system
Authors: Stone D , David A , Bolognani F , Lowenstein PR , Castro MG
Published: December 2004

The transfer of genetic material into endocrine cells and tissues, both in vitro and in vivo, has been identified as critical for the study of endocrine mechanisms and the future treatment of endocrine disorders. Classical methods of gene transfer, such as transfection, are inefficient and limited mainly to delivery into actively proliferating cells in vitro. The development of viral vector gene delivery systems is beginning to circumvent these initial setbacks. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. As different viral vector systems have their own unique advantages and disadvantages, they each have applications for which they are best suited. This review will discuss viral vector systems that have been used for gene transfer into the endocrine system, and recent developments in viral vector technology that may improve their use for endocrine applications - chimeric vectors, viral vector targeting and transcriptional regulation of transgene expression.
The effect of opioid antagonism and environmental restriction on plasma oxytocin and vasopressin concentrations in parturient gilts
Authors: Jarvis S , Lawrence AB , McLean KA , Chirnside J , Deans LA , Calvert SK , Gilbert CL , Goode JA , Forsling ML
Published: December 2004

Oxytocin plays an important role at parturition due to its involvement in uterine contractions, foetal expulsion and the onset of maternal behaviour. The role of the related neurohypophysial hormone, vasopressin, is less clear; however, there is some evidence that it is also involved in maternal behaviour and its role in osmotic regulation is well established. The aim of this study was to investigate the inhibitory effects of endogenous opioids on these hormones during the expulsive phase of parturition in the pig, and to examine how opioid restraint interacts with environmental restriction. The subjects of this study were 31 Large Whitex Landrace primiparous sows (gilts). An indwelling jugular catheter was implanted under general anaesthesia at 12 days before the expected parturition day (EPD). From 5 days before the EPD 15 of the gilts were individually housed in a restrictive parturition crate without straw and 16 were individually housed in a straw-bedded pen. Blood samples were taken with increasing frequency towards and during parturition through a catheter extension to reduce disturbance. At 7.5 min after the birth of the first piglet half of the gilts in each environment received a dose of the opioid receptor antagonist naloxone (1 mg/kg, i.v.) with the remaining gilts receiving saline as a control. Overall, there was no effect of environment on either circulating oxytocin or vasopressin. However, both oxytocin and vasopressin were inhibited by endogenous opioids during the expulsive phase. The inhibitory effects of opioids on these hormones did not appear to have any adverse effects on the progress of parturition as judged by cumulative piglet birth intervals. The regulation of the opioid inhibition of oxytocin and vasopressin during parturition is discussed in relation to other neurotransmitters and whether opioid inhibition of these neurohypophysial hormones is part of the 'normal' physiological response to parturition or whether it is stress-induced.
Diazepam-binding inhibitor/acyl-CoA-binding protein mRNA and peripheral benzodiazepine receptor mRNA in endocrine and immune tissues after prenatal diazepam exposure of male and female rats
Authors: Burgi B , Lichtensteiger W , Schlumpf M
Published: December 2004

Peripheral benzodiazepine (BDZ) receptor (PBR) and diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP) characterized as a ligand at central BDZ receptors, at PBR with involvement in the regulation of steroidogenesis, and as an intracellular acyl-CoA transporter, are both known to interact with BDZ in adult systems. We investigated their expression after prenatal exposure to BDZ. Diazepam (1.25 mg/kg per day s.c.) was administered to time-pregnant Long Evans rats from gestational day (GD) 14 to 20. Expression of mRNAs encoding for PBR and for DBI/ACBP was studied in the same animals with (33)P-labeled 60 mer oligonucleotides (oligos) by in situ hybridization at GD20, and with (32)P-labeled oligos by Northern blot in steroidogenic and immune organs at postnatal day (PN) 14 and in adult offspring. Prenatal diazepam increased DBI/ACBP mRNA expression in male fetal adrenal and in fetal and PN14 testis. Thymus exhibited increased DBI/ACBP mRNA in male fetuses and in adult female offspring, and reduced organ weight at PN14 in both sexes. In female spleen, an increase in DBI/ACBP mRNA and a decrease in PBR mRNA was seen at PN14. Apart from the finding in spleen, no drug-induced changes in PBR mRNA were observed. The effects of prenatal diazepam were superimposed on treatment-independent sex differences in DBI/ACBP mRNA and PBR mRNA expression. Our data indicate that expression of DBI/ACBP mRNA in steroidogenic and immune organs can be affected by exposure to BDZ during ontogeny, while PBR mRNA expression appears to be less sensitive. They further reveal marked sex differences in the developmental patterns of the two proteins during pre- and postpubertal ontogeny.
Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment by capillary electrophoresis
Authors: Arcelloni C , Falqui L , Martinenghi S , Stabilini A , Pontiroli AE , Paroni R
Published: December 2004

The aim of this study was to compare the metabolic pathway to mature insulin through the intermediate forms (32-33 split, 65-66 split, des31,32 and des64,65) in human or murine cells engineered for the release of wild-type human proinsulin and in a genetically mutated one, in the search for a new approach for an insulin-dependent diabetes mellitus cure by gene therapy. Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and NIH3T3) were transduced either with a retroviral vector coding for wild-type proinsulin or for a genetically mutated one, carrying cleavage sites sensitive to furin. The pattern of all the proinsulin cleavage products released into the cell culture supernatants was analyzed by capillary electrophoresis. All the cells transduced with the wild-type gene released intact proinsulin. HepG2 released a considerable amount of 65-66 split and des64,65, while primary myoblasts released all the intermediate forms and a limited amount of mature insulin. All the cells transduced with a furin-sensitive proinsulin gene released a higher amount of mature insulin (23-59% conversion yield) than the cells expressing wild-type proinsulin, whereas the total insulin was nearly constant. Only primary cells released all the cleavage products. Screening a wide variety of non-endocrine cells has revealed a large difference in the processing and release of immature and mature insulin forms, pointing to human hepatic cells as the most efficacious. Capillary electrophoresis provided on-line and in a single run a complete overview of the proinsulin metabolic pathway in different cells.
Tissue-specific regulation of gastrin-releasing peptide synthesis, storage and secretion by oestrogen and progesterone
Authors: Whitley JC , Giraud AS , Mahoney AO , Clarke IJ , Shulkes A
Published: December 2004

In the ovine endometrium, dramatic increases in gastrin-releasing peptide (GRP) mRNA and immunoreactivity are observed during the luteal regression phase of the oestrous cycle (24-fold) and during pregnancy (at least 150-fold). This study sought to determine whether oestrogen and/or progesterone were responsible for the temporal regulation of GRP observed in the uterus. Ovariectomized sheep were divided into four groups (n=4), as follows: 1, untreated; 2, given subcutaneous and intravaginal progesterone implants; 3, given subcutaneous oestrogen implants; and 4, treated with both oestrogen and progesterone. After 10 days, the animals were sacrificed and plasma, pituitary and endometrium were obtained. A fifth group of sheep with intact ovaries was included. Analysis of endometrial GRP-immunoreactivity (GRP-ir) revealed a twofold drop for groups treated with oestrogen, progesterone or both hormones. A dramatic reduction in endometrial GRP mRNA was o! bserved in the group treated with both hormones. GRP-ir was measured in whole pituitaries and found to vary greatly (1.7-53.7 pmol/g tissue) within all groups of ovariectomized animals. There were no significant differences between any of the five groups. A significant reduction in circulating GRP-ir was observed after 10 days of treatment with either oestrogen or progesterone. These studies demonstrate that, in sheep, the synthesis, storage and secretion of GRP are differentially affected by oestrogen and progesterone. Regulation appears to be tissue specific since GRP content in the pituitary is unchanged by oestrogen or progesterone whereas GRP expression in the endometrium is inhibited. Changes in GRP mRNA expression did not correlate with changes in endometrial expression of mRNA for oestrogen receptor alpha, oestrogen receptor beta and the progesterone receptor. This study is the first reported demonstration that expression of the GRP gene can be influenced by the presence of ovarian steroids, with the conclusion that oestrogen and/or progesterone act as negative regulators of endometrial GRP expression.
Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats
Authors: Ramos S , Goya L , Alvarez C , Martin MA , Pascual-Leone AM
Published: December 2004

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.
Retinoid nutritional status differently affects the expression of Japanese quail retinoic acid receptor-beta isoform transcripts in a tissue-specific manner
Authors: Fu ZW , Kubo T , Sugahara K , Noguchi T , Kato H
Published: December 2004

We investigated the effects of vitamin A (VA) nutritional status on the levels of expression of retinoic acid (RA) receptor-beta (RARbeta) gene in the various tissues of Japanese quail. VA deficiency caused a significant decrease in the mRNA levels of brain, liver, heart, lung and kidney RARbeta2/beta4, whereas no change was observed in the level of testis RARbeta2 transcript. In contrast, reduction in the RARbeta1 transcript caused by VA depletion was observed only in the lung, remaining unchanged in the other tissues. The administration of RA to the VA-deficient quail rapidly induced the expression of RARbeta2/beta4 mRNAs in all the tissues examined, but RA increased the expression of RARbeta1 transcript in the liver, heart, lung and kidney at a lower magnitude. RA could not change the expression of the brain RARbeta1 transcript, while it induced the expression of the testis RARbeta1 mRNA in a temporal way. These results clearly indicate that VA nutritional status differently regulates the expression of RARbeta1 and RARbeta2/beta4 transcripts in a tissue-specific manner.
Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis
Authors: Siffroi-Fernandez S , Delom F , Nlend MC , Lanet J , Franc JL , Giraud A
Published: December 2004

Thyroglobulin (Tg) binds to cell surfaces through various binding sites of high, moderate and low affinity. We have previously shown that binding with low to moderate affinity is pH dependent, selective, but not tissue specific. To identify the regions of Tg involved in this cell surface binding, we studied the binding of (125)I-labeled cyanogen bromide peptides from human Tg to cell surfaces of thyroid cells (inside-out follicles) and of CHO cells. Electrophoretic analysis of cell homogenates after binding of native or of reduced and alkylated (125)I-labeled peptides showed that three peptides, P1, P2 and P3, were always associated with the cells. Sequence analysis allowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 to Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly (P2 and P3-2) located in the C-terminal domain of Tg homologous with acetylcholinesterase. The smallest peptides, P1 and P2, were purified by preparative electrophoresis. They both displayed strong binding properties towards cell surfaces. Inhibition experiments of (125)I-labeled Tg binding by P1 or P2 indicated that they were involved in Tg binding to cell surfaces. All the other peptides tested for their binding abilities were either not or only poorly involved in Tg binding to cell surfaces, which suggested that P1 and P2 are major Tg sites of binding to cell surfaces. These two peptides are not involved in the binding of Tg to the known Tg 'receptors' described in the literature, to which recycling, transcytosis and regulation functions have been ascribed. Thus they are potential tools to identify cell surface components involved in the process of Tg endocytosis leading to lysosomal degradation.
GH-releasing peptide-6 overcomes refractoriness of somatotropes to GHRH after feeding
Authors: McMahon CD , Chapin LT , Radcliff RP , Lookingland KJ , Tucker HA
Published: December 2004

After a meal, somatotropes are temporarily refractory to growth hormone-releasing hormone (GHRH), the principal hormone that stimulates secretion of growth hormone (GH). Refractoriness is particularly evident when free access to feed is restricted to a 2-h period each day. GH-releasing peptide-6 (GHRP-6), a synthetic peptide, also stimulates secretion of GH from somatotropes. Because GHRH and GHRP-6 act via different receptors, we hypothesized that GHRP-6 would increase GHRH-induced secretion of GH after feeding. Initially, we determined that intravenous injection of GHRP-6 at 1, 3 and 10 microg/kg body weight (BW) stimulated secretion of GH in a dose-dependent manner. Next, we determined that GHRP-6- and GHRH-induced secretion of GH was lower 1 h after feeding (22.5 and 20 ng/ml respectively) than 1 h before feeding (53.5 and 64.5 ng/ml respectively; pooleds.e.m.=8.5). However, a combination of GHRP-6 at 3 microg/kg BW and GHRH at 0.2 microg/kg BW synergistically induced an equal and massive release of GH before and after feeding that was fivefold greater than GHRH-induced release of GH after feeding. Furthermore, the combination of GHRP-6 and GHRH synergistically increased release of GH from somatotropes cultured in vitro. However, it was not clear if GHRP-6 acted only on somatotropes or also acted at the hypothalamus. Therefore, we wanted to determine if GHRP-6 stimulated secretion of GHRH or inhibited secretion of somatostatin, or both. GHRP-6 stimulated secretion of GHRH from bovine hypothalamic slices, but did not alter secretion of somatostatin. We conclude that GHRP-6 acts at the hypothalamus to stimulate secretion of GHRH, and at somatotropes to restore and enhance the responsiveness of somatotropes to GHRH.
Platelet-derived growth factor (PDGF) and PDGF receptors in rat corpus cavernosum: changes in expression after transient in vivo hypoxia
Authors: Aversa A , Basciani S , Visca P , Arizzi M , Gnessi L , Frajese G , Fabbri A
Published: December 2004

Platelet-derived growth factor (PDGF) overactivity has been implicated in atherosclerosis and several fibrotic conditions including lung and kidney fibrosis, liver cirrhosis and myelofibrosis. Low oxygen tension (hypoxia) is a known stimulus for transcriptional induction of PDGF ligand and receptor genes in different tissues. We studied the expression and localization of PDGF-A, PDGF-B, and PDGF receptor (PDGFR)-alpha and -beta subunits in adult rat isolated corpus cavernosum (CC) under generalized transient hypoxia (pO(2) 10%) in comparison with normoxic conditions. Semi-quantitative RT-PCR analysis of mRNA extracted from rat penis showed higher amounts of PDGF-A, PDGF-B and PDGFR-beta mRNA transcripts in hypoxic versus normoxic animals. The immunohistochemical analysis showed that the localization of PDGF subunits and PDGFR-beta was confined to the cytoplasm of the perivascular smooth muscle cells, endothelium and trabecular fibroblasts. Our findings indicate that transient low oxygen tension induces PDGF overexpression in rat CC, which in the long term may lead to an increase of connective tissue production. We suggest that a local impairment of the PDGF/PDGFR system may contribute to CC fibrosis, which is an established cause of erectile dysfunction in man.
p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts
Authors: Tokuda H , Kozawa O , Miwa M , Uematsu T
Published: December 2004

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.
Co-administration of finasteride and the pure anti-oestrogen ICI 182,780 act synergistically in modulating the IGF system in rat prostate
Authors: Huynh H , Alpert L , Alaoui-Jamali MA , Ng CY , Chan TW
Published: December 2004

Prostate cancer is the most diagnosed invasive malignancy in males. Androgens and oestrogens have been implicated in the pathogenesis of prostate cancer. We report herein that the pure anti-oestrogen ICI 182,780 (ICI) reduces Ki-67 labelling index and IGF-I receptor levels in rat prostate. Increase of IGF-I mRNA and IGF-binding protein 3 (IGFBP-3) accumulation occur without any effect on prostate weight. Finasteride significantly decreases prostate weight and inhibits IGF-I gene expression. IGFBP-3 mRNA, Akt and phospho-Akt are not affected by finasteride. Co-administration of ICI plus finasteride reduces prostate weight by approximately 50% and causes acinar dilation with decreased luminal epithelial cell thickness. The acinar epithelial cells became atrophic and inactive with minimal cytoplasm. We also demonstrate a synergistic effect of ICI and finasteride on induction of IGFBP-3 accumulation and inhibition of Akt phosphorylation. Because the IGF and IGFBP-3 system plays an important role in prostate epithelial cell proliferation, apoptosis and tumour progression, the inhibitory effects of finasteride and ICI on IGF system may contribute to their anti-proliferative activity. These observations support a potential use of ICI in conjunction with finasteride in the prevention and/or treatment of prostate cancer.