bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal exposure to estrogen differentially alters estrogen receptor alpha and beta mRNA expression in rat testis during postnatal development
Authors: Tena-Sempere M , Navarro J , Pinilla L , Gonzalez LC , Huhtaniemi I , Aguilar E
Published: December 2004

The biological actions of estrogens on target cells are mediated by two nuclear receptors: the estrogen receptor (ER) alpha and the recently characterized ER beta. In the male rat, the physiological role of estrogens involves multiple actions, from masculinization of brain areas related to reproductive function and sexual behavior to regulation of testicular development and function. Paradoxically, however, administration of high doses of estrogen during the critical period of neonatal differentiation results in an array of defects in the reproductive axis that permanently disrupt male fertility. The focus of this study was to characterize the effects and mechanism(s) of action of neonatal estrogenization on the pattern of testicular ER alpha and beta gene expression during postnatal development. To this end, groups of male rats were treated at day 1 of age with estradiol benzoate (500 microg/rat), and testicular ER alpha and ER beta mRNA levels were assayed by semi-quantitative RT-PCR from the neonatal period until puberty (days 1-45 of age). Furthermore, the expression of androgen receptor (AR) mRNA was evaluated, given the partially overlapping pattern of tissue distribution of ER alpha, ER beta and AR messages in the developing rat testis. In addition, potential mechanisms for neonatal estrogen action were explored. Thus, to discriminate between direct effects and indirect actions through estrogen-induced suppression of serum gonadotropins, the effects of neonatal estrogenization were compared with those induced by blockade of gonadotropin secretion with a potent LHRH antagonist in the neonatal period. Our results indicate that neonatal exposure to estrogen differentially alters testicular expression of alpha and beta ER messages: ER alpha mRNA levels, as well as those of AR, were significantly decreased, whereas relative and total expression levels of ER beta mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by LHRH antagonist treatment. It is concluded that the effect of estrogen on the expression levels of ER alpha and beta mRNAs probably involves a direct action on the developing testis, and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period.
Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs
Authors: Pelletier G , Labrie C , Labrie F
Published: December 2004

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.
Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary
Authors: Garces D , Mariana JC , Blanc MR , Knight PG , Monniaux D , Collet A , Pisselet C , Fontaine J , Poirier JC
Published: December 2004

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.
Comparison of the effects of add-back therapy with various natural oestrogens on bone metabolism in rats administered a long-acting gonadotrophin-releasing hormone agonist
Authors: Wang Y , Yano T , Kikuchi A , Yano N , Matsumi H , Ando K , Kasai Y , Watanabe M , Okagaki R , Osuga Y , Taketani Y
Published: December 2004

The hypoestrogenic state induced by gonadotrophin-releasing hormone agonist (GnRHa) has been shown to be effective in the treatment of oestrogen-dependent disorders but to induce bone loss. Adding back low doses of oestrogen in GnRHa therapy has been proposed to prevent bone loss. The purpose of this study is to assess the efficacy of add-back therapy with different natural oestrogens such as oestrone (OE(1)), oestradiol (OE(2)) and oestriol (OE(3)). Three-month-old female rats (250 g) were subcutaneously administered microcapsules of leuprorelin acetate in doses of 1 mg/kg of body weight every 4 weeks. GnRHa therapy lasted 16 weeks, and pellets of OE(1), OE(2) or OE(3) (0.5 mg/pellet, 60 day release), as an add-back agent, were implanted at 8 weeks of treatment. At the end of treatment, GnRHa alone decreased bone mineral density of the femur and lumbar vertebrae, and increased serum levels of bone metabolic markers such as alkaline phosphatase and osteocalcin levels. As for cancellous bone histomorphometry, GnRHa decreased bone volume while it increased osteoid volume, osteoid surface, eroded surface, mineral apposition rate and bone formation rate. All the oestrogens tested prevented these changes caused by GnRHa therapy. GnRHa induced a significant increase in body weight and a marked reduction in uterine weight, which was not observed in OE(1) or OE(2) add-back group. Body weight and uterine weight of the OE(3) add-back group were the same as those of the GnRHa group. These findings indicate that GnRHa induces high turnover bone loss which can be prevented by concomitant administration of natural oestrogens such as OE(1), OE(2) and OE(3) to the same extent. In addition, OE(3) is unique in that it is much less effective than OE(1) and OE(2) in blocking body weight gain and in promoting growth of uterine tissues. Because of its tissue-selective actions, OE(3) could be considered as one of the most appropriate oestrogens used for GnRHa add-back therapy.
Vascular endothelial growth factor in the rat pituitary: differential distribution and regulation by estrogen
Authors: Ochoa AL , Mitchner NA , Paynter CD , Morris RE , Ben-Jonathan N
Published: December 2004

Vascular endothelial growth factor (VEGF), an endothelial cell mitogen and permeability factor, participates in tumor angiogenesis, but less is known about its regulation or function in normal vascular homeostasis. In the uterus, which undergoes cyclic changes in its vasculature, VEGF is induced by estrogen. Since the pituitary gland contains highly permeable capillaries and is estrogen-responsive, our objectives were to localize VEGF expression within the pituitary and to determine whether it is regulated by estrogen in both the pituitary and the somatolactotrope cell line, GH(3). Ovariectomized rats were injected with estradiol, and pituitaries and uteri were subjected to in situ hybridization or quantitative reverse transcription-polymerase chain reaction (RT-PCR). VEGF expression was strong and punctate in the neural lobe, weaker and diffuse in the anterior lobe and undetectable in the intermediate lobe. Two VEGF isoforms, 164 and 120, were detected in all tissues. In the posterior pituitary, VEGF expression was 3- to 6-fold higher than in the anterior pituitary or uterus and was unaltered by estrogen. In contrast, anterior pituitary VEGF was induced by estrogen within 1 h, peaked at 3 h, and returned to basal levels by 24 h. Similar dynamics, albeit 10-fold higher, were seen in the uterus. Translated VEGF proteins were detected by Western blot in both the anterior pituitary and uterus. GH(3) cells also showed a dose- and time-dependent induction of VEGF expression by estrogen. In conclusion: (1) VEGF expression is higher in the neural lobe than in the anterior lobe and is undetectable in the intermediate lobe, (2) the expression of VEGF164 and VEGF120 is rapidly upregulated by estrogen in the anterior pituitary but is unchanged in the posterior pituitary, and (3) the pituitary lactotrope cell line, GH(3), also increases VEGF expression in response to estradiol.
Aldosterone mediates angiotensin II-stimulated rat vascular smooth muscle cell proliferation
Authors: Xiao F ,   Puddefoot, Vinson GP
Published: December 2004

Aldosterone, possibly locally generated, has been suggested to have a role in potentiating angiotensin II (AII)-stimulated hypertrophy of cultured vascular smooth muscle cells. To examine the possibility that aldosterone may mediate the proliferative actions of AII, rat aortic smooth muscle cells (RASMCs) in culture were treated with AII in the presence and absence of the specific AII type 1 receptor (AT1) antagonist, losartan, and aldosterone was assayed in culture medium extracts by radioimmunoassay. AII significantly enhanced aldosterone formation (at 10(-8) M: 123.8+/-14.85 vs control 71. 28+/- 8.71 fmol/10(5) cells, P<0.05; at 10(-7) M: 172.38+/-33.44, P<0.05), but not in the presence of losartan (at 10(-8) M: 53. 71+/-18.73, P>0.05; at 10(-7) M: 89.68+/-25.05, P>0.05). In other studies, the reverse transcriptase-polymerase chain reaction, performed on RNA extracted from RASMCs using aldosterone synthase (CYP11B2) specific primers, gave a single band of about 268 bp, consistent with that expected for the enzyme. Finally, using [(3)H]methylthymidine uptake as an index of cellular proliferation, tritium incorporation was increased in the AII-treated group at concentrations greater than 10(-10) M. The aldosterone antagonist, spironolactone (10(-5) M), inhibited the incorporation of [(3)H]thymidine into RASMCs stimulated by AII. These results suggest that locally generated aldosterone may mediate the effects of AII, acting via the AT1 receptor, in stimulating RASMC proliferation.
Tissue-specific regulation of thyroid hormone receptor mRNA isoforms and target gene proteins in domestic ducks
Authors: Bishop CM , McCabe CJ , Gittoes NJ , Butler PJ , Franklyn JA
Published: December 2004

Skeletal muscles are important target tissues for thyroid hormone action. The present study examines the influence of thyroid status on muscle growth and tissue-specific expression of thyroid receptor (TR) mRNA isoforms in a commercial strain of the domestic duck (Anas platyrhynchos). Four groups (n=5) of 1-week-old ducklings were rendered either hypothyroid by treatment with methimazole (6 mg 100 g(-1) body mass or 12 mg 100 g(-1) body mass), or hyperthyroid by treatment with methimazole (6 mg 100 g(-1) body mass) in combination with thyroid hormones (5 microg thyroxine (T(4)) and tri-iodothyronine (T(3)) 100 g(-1) body mass or 10 microg T(4) and T(3) 100 g(-1) body mass). Serum and tissue samples (cardiac, pectoralis and semimembranosus leg muscle, liver, pituitary and cerebral cortex) were collected from these four groups, and from a group of untreated controls, at 8 weeks of age. Development of duckling morphology was retarded in methimazole-treated birds compared with that in euthyroid controls, as evidenced by differences in skeletal dimensions, primary feather length, and body and muscle masses. Body mass was lower by 18%, and relative masses of cardiac and pectoralis muscles were lower by 28% and 32% respectively. Heterologous oligonucleotides for TR alpha, TR beta 0, TR beta2 and the housekeeping gene beta-actin were derived from chicken sequences. RT-PCR showed that TR alpha mRNA was expressed in all tissues but was not significantly affected by any of the experimental treatments. TR beta 0 mRNA expression was significantly lower in the leg muscles of ducklings treated with 12 mg methimazole 100 g(-1) body mass (0.109+/-0.047 TR:beta-actin ratio, P<0.05) compared with that in euthyroid controls (0.380+/-0.202), but was unaltered in the pectoralis and cardiac muscles. Expression of TR beta 0 mRNA was significantly higher in pectoralis (by 3.5-fold, P<0. 05), cardiac (by 4.2-fold, P=0.003) and leg (by 4.0-fold, P<0.001) muscles of ducklings treated with thyroid hormones compared with those in euthyroid controls (0.098+/-0.019, 0.822+/-0.297 and 0. 38+/-0.202 TR:beta-actin respectively). Only the pituitary gland expressed significant levels of TR beta 2 mRNA.
Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells
Authors: Kim NH , Jung HH , Cha DR , Choi DS
Published: December 2004

Diabetic nephropathy associated with hyperglycemia is characterized by glomerular hyperfiltration and endothelial dysfunction. Vascular endothelial growth factor (VEGF) is known to be primarily involved in neoangiogenesis and increased endothelial permeability. The purpose of this study was to investigate VEGF expression in response to high glucose in rat cultured mesangial cells and to identify its signal pathway via protein kinase C (PKC). Rat mesangial cells were cultured with different concentrations of glucose: normal (5 mM d-glucose), medium (15 mM d-glucose) and high (30 mm d-glucose). Calphostin-C as a PKC inhibitor and phorbol myristate acetate (PMA) as a PKC downregulator were instillated into culture media to evaluate the role of PKC in mediating the glucose-induced increase in VEGF expression. High glucose increased expression of VEGF at the mRNA and protein levels, identified by semi-quantitative RT-PCR and western blotting, within 3 h and in a time- and glucose concentration-dependent manner. Calphostin-C and PMA inhibited glucose-induced increases in VEGF expression at the mRNA and protein levels. In conclusion, high glucose can directly increase VEGF expression in rat mesangial cells via a PKC-dependent mechanism. These results suggest that VEGF could be a potential mediator of glomerular hyperfiltration and proteinuria in diabetic nephropathy.
Parathyroid hormone-related protein(1-34) in gestational fluids and release from human gestational tissues
Authors: Farrugia W , Ho PW , Rice GE , Moseley JM , Permezel M , Wlodek ME
Published: December 2004

Parathyroid hormone-related protein (PTHrP) is present in fetal and gestational tissues, in which its proposed roles include stimulation of epithelial growth and differentiation, vasodilatation of the uteroplacental vasculature, relaxation of uterine muscle and stimulation of placental calcium transport. The aim of this study was to determine whether the release of PTHrP from gestational tissue explants was tissue specific. In addition, PTHrP concentrations were measured in maternal plasma, umbilical artery and vein plasma, and amniotic fluid from term, uncomplicated pregnancies before the onset of labour. PTHrP was detected in low concentrations in the mother, fetus and placental tissue. Amniotic fluid had ten times the PTHrP concentration compared with that in the maternal or fetal circulations. Using late pregnant human gestational tissues in an in vitro explant system, we found that amnion over placenta, choriodecidua, reflected amnion, and placenta released PTHrP into culture medium in progressively greater amounts over 24 h (P<0.05). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation media. After 24 h incubation, the fetal membranes released significantly (P<0.05) greater amounts of PTHrP than did the placenta (placenta 3. 7+/-0.5 pmol PTHrP/g protein). Amnion over placenta released significantly more PTHrP (139.3+/- 43.1 pmol PTHrP/g protein) than did reflected amnion (29.0+/-8.3 pmol PTHrP/g protein) (P<0.05). This study unequivocally demonstrated that human gestational tissues release PTHrP and it was concluded that the main contributors to PTHrP in amniotic fluid were the human fetal membranes, particularly amnion over placenta. Fetal membrane-derived and amniotic fluid PTHrP are proposed to have stimulatory effects on epithelial growth and differentiation in fetal lung, gut, skin and hair follicles and paracrine effects on placental vascular tone and calcium transport.
Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods
Authors: Stridsberg M , Angeletti RH , Helle KB
Published: December 2004

Chromogranin A (CgA) and chromogranin B (CgB) are acidic proteins stored in and released from hormone granules in endocrine and neuroendocrine tissue. The chromogranins are postulated to serve as pro-hormones to generate biologically active peptides, which may influence hormonal release and vascular functions or have antibacterial functions. Although N-terminal and C-terminal regions show some species amino acid homology, the chromogranins as a whole display considerable interspecies differences, which prevents their use in comparative studies of biological functions. We present four new radioimmunoassays for the measurement of defined N-terminal regions of CgA and CgB. A new radioimmunoassay for measurement of intact bovine CgA has also been developed. With these assays and two previously published ones, we have compared the cross-reactivity of chromogranins from man, cattle, sheep, goat, pig and horse and compared adrenomedullar content and serum levels of CgA from these species. We have also studied the influence of peptide concentrations and the ionic strength of the mobile phase on molecular weight estimations. Assays with antibodies directed against the N-terminal parts of CgA and CgB showed sufficient interspecies cross-reactivity to allow comparative quantification of the circulating levels in man, cattle, sheep, goat, pig and horse. Assays measuring the intact human or bovine CgA were not suitable for comparative purposes in samples from sheep, goat, pig and horse. Molecular interactions between vasostatin immunoreactive material and intact bovine CgA were demonstrated in gel permeation studies, suggesting that conclusions about the degree of N-terminal processing from elution profiles should be made with caution. Reliable interspecies comparison of chromogranins is difficult, but measurements with region-specific assays may be helpful to study concentrations of chromogranins and chromogranin-related peptides.
Possible relationship between changes in islet neogenesis and islet neogenesis-associated protein-positive cell mass induced by sucrose administration to normal hamsters
Authors: Del Zotto H , Massa L , Rafaeloff R , Pittenger GL , Vinik A , Gold G , Reifel-Miller A , Gagliardino JJ
Published: December 2004

The possible relationship between changes in islet cell mass and in islet neogenesis-associated protein (INGAP)-cell mass induced by sucrose administration to normal hamsters was investigated. Normal hamsters were given sucrose (10% in drinking water) for 5 (S8) or 21 (S24) weeks and compared with control (C) fed hamsters. Serum glucose and insulin levels were measured and quantitative immunocytochemistry of the endocrine pancreas was performed. Serum glucose levels were comparable among the groups, while insulin levels were higher in S hamsters. There was a significant increase in beta-cell mass (P<0.02) and in beta-cell 5-bromo-2'-deoxyuridine index (P<0.01), and a significant decrease in islet volume (P<0.01) only in S8 vs C8 hamsters. Cytokeratin (CK)-labelled cells were detected only in S8 hamsters. INGAP-positive cell mass was significantly larger only in S8 vs C8 hamsters. Endocrine INGAP-positive cells were located at the islet periphery ( approximately 96%), spread within the exocrine pancreas ( approximately 3%), and in ductal cells (<1%) in all groups. INGAP positivity and glucagon co-localization varied according to topographic location and type of treatment. In C8 hamsters, 49.1+/-6. 9% cells were INGAP- and glucagon-positive in the islets, while this percentage decreased by almost half in endocrine extra-insular and ductal cells. In S8 animals, co-expression increased in endocrine extra-insular cells to 36.3+/-9.5%, with similar figures in the islets, decreasing to 19.7+/-6.9% in ductal cells. INGAP-positive cells located at the islet periphery also co-expressed CK. In conclusion, a significant increase of INGAP-positive cell mass was only observed at 8 weeks when neogenesis was present, suggesting that this peptide might participate in the control of islet neogenesis. Thus, INGAP could be a potentially useful tool to treat conditions in which there is a decrease in beta-cell mass.
Leptin expression in offspring is programmed by nutrition in pregnancy
Authors: Eckert JE , Gatford KL , Luxford BG , Campbell RG , Owens PC
Published: December 2004

Birth weight is a determinant of blood leptin concentrations in adults. Since nutrition during pregnancy can affect birth weight, the hypothesis that feed intake during pregnancy alters leptin expression in progeny was examined. Leptin mRNA was measured in subcutaneous adipose tissue and leptin protein was measuredin blood plasma from 59 day old female pigs whose mothers were fed at the same restricted rate except that half were permitted to consume 35% more feed during the second quarter of pregnancy. Leptin mRNA abundance in adipose tissue (P=0.015) and plasma leptin concentration (P=0.01) were higher in progeny from mothers provided with more feed. Body weight at birth was negatively correlated with the abundance of leptin mRNA in subcutaneous fat at 59 days of age (P=0.01). This study shows for the first time that maternal nutrition during pregnancy programs postnatal leptin expression in offspring.
Multiple endocrine neoplasia type 1 (MEN1) gene mutations in a subset of patients with sporadic and familial primary hyperparathyroidism target the coding sequence but spare the promoter region
Authors: Karges W , Jostarndt K , Maier S , Flemming A , Weitz M , Wissmann A , Feldmann B , Dralle H , Wagner P , Boehm BO
Published: December 2004

Germ line mutations of the multiple endocrine neoplasia type 1 (MEN1) tumour suppressor gene cause MEN1, a rare familial tumour syndrome associated with parathyroid hyperplasia, adenoma and hyperparathyroidism (HP). Here we investigated the role of the MEN1 gene in isolated sporadic and familial HP. Using RT-PCR single-strand conformational polymorphism screening, somatic (but not germ line) mutations of the MEN1 coding sequence were identified in 6 of 31 (19.3%) adenomas from patients with sporadic primary HP, but none in patients (n=16) with secondary HP due to chronic renal failure. MEN1 mutations were accompanied by a loss of heterozygosity (LOH) for the MEN1 locus on chromosome 11q13 in the adenomas as detected by microsatellite analysis. No DNA sequence divergence within the 5' region of the MEN1 gene, containing the putative MEN1 promoter, was detectable in HP adenomas. Clinical characteristics were not different in HP patients with or without MEN1 mutation. Heterozygous MEN1 gene polymorphisms were identified in 9.6% and 25% of patients with primary and secondary HP respectively. In a large kindred with familial isolated familial HP, MEN1 germ line mutation 249 del4 and LOH was associated with the HP phenotype and a predisposition to non-endocrine malignancies. We suggest that the bi-allelic somatic loss of MEN1 wild-type gene expression is involved in the pathogenesis of a clinically yet undefined subset of sporadic primary HP adenomas. MEN1 genotyping may further help define the familial hyperparathyroidism-MEN1 disease complex, but it seems dispensable in sporadic primary HP.
Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes
Authors: Matsumoto T , Tsurumoto T , Goldring MB , Shindo H
Published: December 2004

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.
Expression of proliferating cell nuclear antigen in luminal epithelium during the growth and regression of rat uterus
Authors: Lai MD , Lee LR , Cheng KS , Wing LY
Published: December 2004

Proliferating cell nuclear antigen (PCNA), a processivity factor of DNA synthesis, has often been used as a marker that reveals proliferating cells. However, it also plays a role other than in DNA replication. The aim of this study was to examine the relationship between the expression of PCNA and cell proliferation, and also its relation to cell death in the uterine epithelium under various hormonal conditions. Rats with regular estrous cycles were killed at various stages of the cycle, and their uteri were removed for the detection of PCNA and apoptosis by immunohistochemical and terminal deoxynucleotidyl transferase-mediated nick end-label staining respectively. There was an inverse relationship between the expression of PCNA and apoptosis in the uterine epithelium during the estrous cycle. From diestrus to proestrus, the expression of PCNA increased, and few apoptotic cells were detected in the luminal epithelium. However, at estrus, apoptosis occurred markedly, and the expression of PCNA disappeared. To study further the effects of estrogen on PCNA expression and cell growth in the uterus, rats were ovariectomized and then implanted s.c. with estrogen capsules 2 weeks later. In ovariectomized rats, only a few PCNA-positive cells were observed in the uterine epithelium. After estrogen treatment, PCNA was expressed strongly in the luminal and glandular epithelia. In these rats, the removal of estrogen capsules resulted in apoptotic death and surprisingly strong PCNA expression in the cells of luminal epithelium. Our results demonstrate that PCNA is expressed not only in the estrogen-stimulated uterine growth, but also in the processes of regression induced by the withdrawal of estrogen. Although the expression of PCNA has been reported to represent cell proliferation, our results implicate functions other than cell replication for PCNA in the uterus.
Factors affecting production of luciferase and epitope-tagged IGF-I in porcine muscle after DNA injection
Authors: Everett RS , Gerrard DE , Grant AL
Published: December 2004

Direct DNA injection into porcine skeletal muscle was investigated as an approach for studying roles of locally produced IGF-I on IGF-binding protein (IGFBP) production. To determine parameters for maximal reporter gene expression, and to investigate the effects of dose, time and weaning on exogenous DNA expression, plasmid DNA encoding firefly luciferase under control of a constitutive promoter and enhancer was injected in skeletal muscle of pigs. Results indicate that injected DNA does not migrate beyond 9 mm from injection sites and that 100 microg DNA injections resulted in optimal luciferase activity. Maximum amounts of recombinant protein were observed 3 days after injection, and were reduced by weaning. Using these data, a second DNA injection study was performed using plasmid DNA containing a cDNA insert for epitope-tagged insulin-like growth factor-I (TIGF-I). Significant quantities of TIGF-I were detected by ELISA and confirmed by western blotting. Both IGFBP-2 and IGFBP-2 mRNA were increased in treated muscle compared with controls. We conclude that increased expression of IGF-I in muscle results in increased IGFBP-2. Furthermore, these data indicate that this in vivo approach of gene transfer results in biologically active recombinant protein production in porcine skeletal muscle, and provides an excellent in vivo model for studying the autocrine and (or) paracrine effects of locally produced growth factors in skeletal muscle.
Two estrogen receptors expressed in the teleost fish, Sparus aurata: cDNA cloning, characterization and tissue distribution
Authors: Socorro S , Power DM , Olsson PE , Canario AV
Published: December 2004

Estrogen is an essential hormone for many reproductive and non-reproductive functions. The function of estrogen in the reproductive cycle of seabream (Sparus aurata), a protandrous hermaphrodite teleost fish, is complex but it is understood to be involved in sex inversion, a process that occurs in some individuals during the second reproductive season. Estrogen action is mediated by two estrogen receptor (ER) subtypes designated alpha and beta. As a step to understanding the mechanisms of estrogen action during natural and induced sex reversal in seabream, we have isolated two cDNAs encoding distinct forms of ER homologous to mammalian ERalpha and ERbeta. The seabream ERalpha clone (sbERalpha1), which was truncated in the A/B domain, corresponded to a variant differing in five amino acids from another recently cloned sbERalpha. The ERbeta clone (sbERbeta) encoded a protein 559 amino acids long and showed only 40% identity to sbERalpha. Northern blot analysis of liver and ovary mRNA indicated the presence of several transcripts of the two receptor subtypes. PCR analysis showed that the two receptors differed in their expression pattern. sbERalpha had a more restricted distribution, occurring mainly in testis, liver and heart, and sbERbeta was present in most tissues, being more abundant in ovary, testis, liver, intestine and kidney. The presence in seabream of two ERs with several ER transcripts and their pattern of distribution are consistent with the widespread effects of estrogen in different tissues.
Interaction between substance P and TRH in the control of prolactin release
Authors: Duvilanski BH , Pisera D , Seilicovich A , del Carmen Diaz M , Lasaga M , Isovich E , Velardez MO
Published: December 2004

Substance P (SP) may participate as a paracrine and/or autocrine factor in the regulation of anterior pituitary function. This project studied the effect of TRH on SP content and release from anterior pituitary and the role of SP in TRH-induced prolactin release. TRH (10(-7) M), but not vasoactive intestinal polypeptide (VIP), increased immunoreactive-SP (ir-SP) content and release from male rat anterior pituitary in vitro. An anti-prolactin serum also increased ir-SP release and content. In order to determine whether intrapituitary SP participates in TRH-induced prolactin release, anterior pituitaries were incubated with TRH (10(-7) M) and either WIN 62,577, a specific antagonist of the NK1 receptor, or a specific anti-SP serum. Both WIN 62,577 (10(-8) and 10(-7) M) and the anti-SP serum (1:250) blocked TRH-induced prolactin release. In order to study the interaction between TRH and SP on prolactin release, anterior pituitaries were incubated with either TRH (10(-7) M) or SP, or with both peptides. SP (10(-7) and 10(-6) M) by itself stimulated prolactin release. While 10(-7) M SP did not modify the TRH effect, 10(-6) M SP reduced TRH-stimulated prolactin release. SP (10(-5) M) alone failed to stimulate prolactin release and markedly decreased TRH-induced prolactin release. The present study shows that TRH stimulates ir-SP release and increases ir-SP content in the anterior pituitary. Our data also suggest that SP may act as a modulator of TRH effect on prolactin secretion by a paracrine mechanism.
Dehydroepiandrosterone prevents lipid peroxidation and cell growth inhibition induced by high glucose concentration in cultured rat mesangial cells
Authors: Brignardello E , Gallo M , Aragno M , Manti R , Tamagno E , Danni O , Boccuzzi G
Published: December 2004

The oxidative stress induced by high glucose concentration contributes to tissue damage associated with diabetes, including renal injury. Dehydroepiandrosterone (DHEA), the major secretory product of the human adrenal gland, has been shown to possess a multi-targeted antioxidant activity which is also effective against lipid peroxidation induced by high glucose. In this study we evaluated the effect of DHEA on the growth impairment which high glucose concentration induces in cultured rat mesangial cells. Primary cultures of rat mesangial cells were grown for 10 days in media containing either normal (i.e. 5.6 mmol/l) or high (i.e. 30 mmol/l) concentrations of glucose, without or with DHEA at different concentrations. The impairment of cell growth induced by high glucose was reversed by 100 nmol/l and 500 nmol/l DHEA, which had no effect on mesangial cells cultured in media containing glucose at the normal physiological concentration (5.6 mmol/l). In high-glucose cultured mesangial cells, DHEA also attenuated the lipid peroxidation, as measured by thiobarbituric acid reactive substances (TBARS) generation and 4-hydroxynonenal (HNE) concentration, and preserved the cellular content of reduced glutathione as well as the membrane Na+/K+ ATPase activity. The data further support the protective effect of DHEA against oxidative damage induced by high glucose concentrations, and bring into focus its possible effectiveness in preventing chronic complications of diabetes.
Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep
Authors: Ehrhardt RA , Slepetis RM , Siegal-Willott J , Van Amburgh ME , Bell AW , Boisclair YR
Published: December 2004

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.
Transfection of human insulin-like growth factor-binding protein 3 gene inhibits cell growth and tumorigenicity: a cell culture model for lung cancer
Authors: Hochscheid R , Jaques G , Wegmann B
Published: December 2004

IGF-I and IGF-II are potent mitogens, postulated to exert autocrine/paracrine effects on growth regulation in human lung cancer. Their proliferative effects are modulated by IGF-binding proteins (IGFBPs), which are found in conditioned medium (CM) of lung cancer cell lines. The biological role of the IGFBPs, which are ontogenetically and hormonally regulated, is not fully understood. Both inhibitory and stimulatory effects on cell growth have been demonstrated. Exogenous IGFBP-3 has been consistently shown to block IGF action, inhibiting cell growth in vitro. In order to evaluate the action of endogenously produced IGFBP-3 on cell growth in lung cancer, we stably transfected the non-small cell lung cancer cell line NCI-H23 with human IGFBP-3 cDNA (resulting in NCI-H23 pOPI3/BP-3) or with the vector pOPI3CAT as control (resulting in NCI-H23 pOPI3CAT). RT-PCR confirmed expression of IGFBP-3-specific mRNA in NCI-H23 pOPI3/BP-3, but not in N! CI-H23 or NCI-H23 pOPI3CAT. Western ligand blot and Western immunoblot analysis of CMs yielded strong signals of the characteristic 40-44 kDa human IGFBP-3 protein in NCI-H23 pOPI3/BP-3. An IGFBP-3 ELISA demonstrated a 20-fold increase in IGFBP-3 protein expression in NCI-H23 pOPI3/BP-3 as compared with NCI-H23. The growth of NCI-H23 pOPI3/BP-3 in serum-containing medium was significantly slower (1.7-fold) than that of NCI-H23 or the vector-transfected control NCI-H23 pOPI3CAT. While the proliferation rate of parental and vector-transfected cells could be stimulated by IGF-I, IGF-II, IGF-I analog Long R(3) IGF-I or insulin, that of NCI-H23 pOPI3/BP-3 could not. Xenotransplantation in nude mice resulted in marked tumor growth after the injection of NCI-H23 or NCI-H23 pOPI3CAT, but absent or minimal growth for the IGFBP-3-transfected cell line. These data suggest that IGFBP-3 is a potent inhibitor of cell growth in human lung cancer c! ell lines and may impair tumorigenicity in vivo.
Compensatory alterations of insulin signal transduction in liver of growth hormone receptor knockout mice
Authors: Dominici FP , Arostegui Diaz G , Bartke A , Kopchick JJ , Turyn D
Published: December 2004

Growth hormone (GH) deficiency is associated with increased sensitivity to insulin, but the molecular mechanisms involved in this association are poorly understood. In the current work, we have examined the consequences of the absence of the biological effects of GH on the first steps of the insulin signaling system in vivo in liver of mice with targeted disruption of the GH receptor/GH binding protein gene (GHR-KO mice). In these animals, circulating insulin concentrations are less than 4 microIU/ml, and glucose concentrations are low, concordant with a state of insulin hypersensitivity. The abundance and tyrosine phosphorylation state of the insulin receptor (IR), the IR substrate-1 (IRS-1), and Shc, the association between IRS-1 and the p85 subunit of phosphatidylinositol (PI) 3-kinase, the IRS-1- and the phosphotyrosine-associated PI 3-kinase in liver were examined. We found that, in liver of GHR-KO mice, the lack of GHR and GH eff! ects is associated with: (1) increased IR abundance, (2) increased insulin-stimulated IR tyrosine phosphorylation, (3) normal efficiency of IRS-1 and Shc tyrosine phosphorylation and (4) normal activation of PI 3-kinase by insulin. These alterations could represent an adaptation to the low insulin concentrations displayed by these animals, and may account for their increased insulin sensitivity.
Quantification and synthesis of cocaine- and amphetamine-regulated transcript peptide (79-102)-like immunoreactivity and mRNA in rat tissues
Authors: Murphy KG , Abbott CR , Mahmoudi M , Hunter R , Gardiner JV , Rossi M , Stanley SA , Ghatei MA , Kuhar MJ , Bloom SR
Published: December 2004

The distribution of cocaine- and amphetamine-regulated transcript peptide (79-102)-like immunoreactivity (CART-LI) was quantified in brain and peripheral tissues of male and female Wistar rats, and male obese (fa/fa) and heterozygous (Fa/+) Zucker rats using a specific RIA. CART-LI tissue levels have not been quantified previously. The assay, using cocaine- and amphetamine-regulated transcript (CART) (79-102) as a standard and radioactive tracer and an antibody to CART (79-102) fragment, detected CART-LI in all brain regions examined, the anterior and posterior pituitary, the spinal cord and throughout the gastrointestinal tract of both male and female Wistar rats. The highest concentrations were found in the hypothalamus, duodenum, anterior pituitary and posterior pituitary (50.6+/-4.4, 26.1+/-4.2, 50.0+/-1.3 and 373.0+/-55.2 pmol/g wet tissue respectively, means+/- s.e.m., n=6-10 male animals). There was no significant variation between the sexes. The concentrations of CART-LI in hypothalami and anterior and posterior pituitaries from fa/fa rats were significantly (P<0002) lower than those of Fa/+ controls (35.9+/-2.1 vs 53.9+/-4.9,<0.6 vs 1.8+/-0.4 and 114+/-9.1 vs 255.5+/- 20.9 pmol/g wet tissue respectively, means+/- s.e.m., n=7). Gel permeation chromatography of regions of rat brain and gastrointestinal tract showed possible differential processing between regions. CART-LI was released from hypothalamic tissue slices in a calcium-dependent fashion by potassium-induced depolarisation. Northern blot analysis detected CART mRNA in the hypothalamus, anterior pituitary, brain stem, cerebellum and spinal cord. The pattern o! f distribution of CART mRNA and CART-LI in various neural and other tissues is in accord with a role for CART as a neurotransmitter.
Parathyroid hormone(1-34) and parathyroid hormone-related protein(1-34) stimulate calcium release from human syncytiotrophoblast basal membranes via a common receptor
Authors: Farrugia W , de Gooyer T , Rice GE , Moseley JM , Wlodek ME
Published: December 2004

The placental syncytiotrophoblast is the site for mineral and nutrient exchange across the maternal-fetal interface. It has been proposed that parathyroid hormone-related protein (PTHrP) is a key factor in the maintenance of a maternal-fetal calcium gradient. Using simultaneously prepared microvillous (maternal facing) and basal (fetal facing) syncytiotrophoblast membranes from term human placentae (n=8), we determined the relative contribution of PTH(1-34), PTHrP(1-34) and PTHrP(67-94) to the regulation of syncytiotrophoblast calcium efflux. The vesicles had correct right-side-out membrane orientation and specific markers validated the fractionation of microvillous and basal membrane vesicles. Calcium efflux was studied by preloading vesicles with calcium-45 in the presence of calcium and magnesium and then incubating the vesicles at 37 degrees C for 15 min with the peptides. In basal membranes, PTHrP(1-! 34) significantly stimulated calcium efflux at a dose of 12.5 nmol/l, whereas PTH(1-34)-stimulated efflux was significant at 50 nmol/l (P<0.05, ANOVA). This efflux was significantly reduced in the presence of the PTH/PTHrP receptor antagonist (PTHrP(7-34)). Midmolecule PTHrP(67-94) had no significant effect on basal membrane calcium efflux. PTH(1-34), PTHrP(1-34) or PTHrP(67-94) had no significant effects on MVM calcium efflux. This study, using the human syncytiotrophoblast in vitro membrane system, demonstrated that PTHrP(1-34) and PTH(1-34) stimulate calcium transport across the basal, but not microvillous, syncytiotrophoblast membrane vesicles, mediated via the PTH/PTHrP receptor.