bluuebunny/crossref_metadata_2025 huggingface dataset:
Persistent gene expression in mouse vagina exposed neonatally to diethylstilbestrol
Authors: Miyagawa S , Suzuki A , Katsu Y , Kobayashi M , Goto M , Handa H , Watanabe H , Iguchi T
Published: December 2004

Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.
Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice
Authors: Lemmen JG , Arends RJ , van Boxtel AL , van der Saag PT , van der Burg B
Published: December 2004

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.
Integrin-mediated action of insulin-like growth factor binding protein-2 in tumor cells
Authors: Schutt BS , Langkamp M , Rauschnabel U , Ranke MB , Elmlinger MW
Published: December 2004

The neoplastic production of the insulin-like growth factor binding protein (IGFBP)-2 often correlates with tumor malignancy and aggressiveness. Since IGFBP-2 contains an RGD motif in its C-terminus, it was hypothesized that this protein may act independently of IGF on tumor cells through integrins. To investigate this, integrin binding, intracellular signaling and the impact of IGFBP-2 on cell adhesion and proliferation were examined in two tumor cell lines. In tracer displacement studies, up to 30% of the added (125)I-hIGFBP-2 specifically bound to the cells. Bound (125)I-hIGFBP-2 was reversibly displaced by IGFBP-2, IGFBP-1 and RGD-(Gly-Arg-Asp)-containing peptides, but not by IGFBP-3, -4, -5, -6 and RGE-(Gly-Arg-Glu)-containing peptides. Blocking with antibodies directed against different integrins and with fibronectin demonstrated that IGFBP-2 cell surface binding is specific for alpha5beta1-integrin. Incubation of IGFBP-2 with equimolar quantities of IGF-I and IGF-II annihilated RGD-specific binding. IGFBP-2 binding at the cell surface led to dephosphorylation of the focal adhesion-kinase (FAK) of up to 37% (P<0.01), and of the p42/44 MAP-kinases of up to 40% (P<0.01). In addition, IGFBP-2 promoted de-adhesion of the cells dose-dependently by up to 30% (P<0.05), and reduced proliferation by 24% (P<0.01). Since one of the cell lines used does not express a functional IGF-I receptor, these data demonstrate that IGFBP-2 can act in an IGF-independent manner, at least in part by an interaction with alpha5beta1-integrin.
Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism
Authors: Baryshev M , Sargsyan E , Wallin G , Lejnieks A , Furudate S , Hishinuma A , Mkrtchian S
Published: December 2004

The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the protein folding and processing capacity of the endoplasmic reticulum (ER). The UPR is induced by the pharmacological agents that perturb ER functions but is also activated upon excessive accumulation of the mutant secretory proteins that are unable to attain correct three-dimensional structure and are thus retained in the ER. Such defects in intracellular protein transport underlie the development of a number of phenotypically diverse inherited pathologies, termed endoplasmic reticulum storage diseases (ERSD). We have studied UPR development in two similar ERSDs, human congenital goiter caused by the C1264R and C1996S mutations in the thyroglobulin (Tg) gene and non-goitrous congenital hypothyroidism in rdw dwarf rats determined by the G2320R Tg mutation. In both cases, these mutations rendered Tg incapable of leaving the ER. A major ER chaperone immunoglobulin-binding protein (BiP), and a novel putative escort chaperone endoplasmic reticulum protein 29 KDa (ERp29) were found to be associated with Tg, which might be interpreted as the contribution of the quality control machinery to the previously shown retention of Tg in the ER. We have extended our earlier observations of ER chaperone induction with the identification of the additional ER (ERp29, ERp72, calreticulin, protein disulfide isomerase (PDI)), cytoplasmic (heat shock protein (HSP)70, HSP90) and mitochondrial (mtHSP70) upregulated chaperones and folding enzymes. Activation of the transcriptional arm of UPR, as judged by the appearance of the spliced (active) form of X-box binding protein (XBP1) and processed activating transcription factor 6 (ATF6) transcription factors was suggested to contribute to the overexpression of the ER chaperones. The processing of ATF6 was observed in both human and rat tissues with Tg mutations. Whereas, in human tissues, weak splicing of XBP1 mRNA was detected only in the C1264R mutant, all rat thyroids including wild-type contained significant amounts of the spliced form of XBP1 as opposed to human liver and rat brain tissues, implying the existence of a previously unknown tissue-specific regulation of XBP1 processing.
The molecular characteristics of polycystic ovary syndrome (PCOS) ovary defined by human ovary cDNA microarray
Authors: Diao FY , Xu M , Hu Y , Li J , Xu Z , Lin M , Wang L , Zhou Y , Zhou Z , Liu J , Sha J
Published: December 2004

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders; it is characterized by polycystic ovaries, hyperandrogenism and chronic anovulation. To obtain a global view of those genes that might be involved in the development of this complex clinical disorder, we used recently developed cDNA microarray technology to compare differential gene expressions between normal human ovary and ovaries from PCOS patients. A total of 9216 clones randomly selected from a commercial human ovary cDNA library were screened. Among them, 290 clones showed differential expressions, including 119 known genes and 100 known or unknown expressed sequence tags (ESTs). Among 119 known genes, 88 were upregulated and 31 downregulated in the PCOS ovary, as compared with normal human ovary. These differentially expressed genes are involved in various biologic functions, such as cell division/apoptosis, regulation of gene expression and metabolism, reflecting the complexity of clinical manifestations of PCOS. The molecular characteristics established from our study will further our understanding of the pathogenesis of PCOS and help us to identify new targets for further studies and for the development of new therapeutic interventions.
IGF-I inhibits apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway in pituitary cells
Authors: Fernandez M , Sanchez-Franco F , Palacios N , Sanchez I , Fernandez C , Cacicedo L
Published: December 2004

In previous studies we demonstrated that IGF-I induces proliferation of pituitary lactotrophs. In addition to its mitotrophic actions, IGF-I is known to prevent apoptosis induced by diverse stimuli in several cell types. In this study, we investigated the action of IGF-I on pituitary cell survival and the intracellular signaling transduction pathway implicated in this effect. Treatment of cultured male rat pituitary cells with IGF-I (10(-7) M) for 24 h prevented pituitary cell death induced by serum deprivation. The protective effect of IGF-I was blocked by phosphoinositide 3-kinase (PI3-kinase) inhibitor, LY294002, but was unaffected by PD98059, which inhibits MAP/ERK kinase (MEK1). IGF-I activation of PI3-kinase induced the phosphorylation and activation of the serine/threonine kinase Akt. Moreover, IGF-I increased the phosphorylation of the pro-apoptotic factor Bad and the levels of the anti-apoptotic protein Bcl-2 through the PI3-kinase pathway in primary pituitary cells.
Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability
Authors: Sellers RS , Luchin AI , Richard V , Brena RM , Lima D , Rosol TJ
Published: December 2004

Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3'-untranslated region (3'-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-beta1 (TGF-beta1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-beta1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-beta1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein-RNA binding studies identified different proteins binding to the 3'-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-beta1-induced changes in PTHrP mRNA isoform expression and stability.
Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase
Authors: Thomas JL , Duax WL , Addlagatta A , Scaccia LA , Frizzell KA , Carloni SB
Published: December 2004

Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent.These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.
A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron
Authors: Steketee Karine , Ziel-van der Made Angelique C J , van der Korput Hetty A G M , Houtsmuller Adriaan B , Trapman Jan
Published: December 2004

We characterized the specifically androgen-regulated gene (SARG), which is expressed in the androgen receptor (AR) and glucocorticoid receptor (GR) positive cell line lymph node carcinoma of the prostate-1F5 (LNCaP-1F5). SARG mRNA expression can be up-regulated by androgens, but not by glucocorticoids. SARG mRNA expression is high in prostate tissue. SARG is composed of four exons and spans a region of 14.5 kbp on chromosome 1q32.2. Transcripts of 5.5, 3.3 and 2.3 kb are the result of alternative polyadenylation. SARG mRNA splice variants lack exon 2 and vary in length of exon 1. The SARG protein has a length of 601 amino acids and is located in the cytoplasm. By screening the 18 kbp genomic sequence flanking the transcription start site we identified the imperfect direct repeat 5′-TGTGCTaacTGTTCT-3′in intron 1 as an active androgen response element (ARE-SARG+4.6). A 569 bp genomic DNA fragment containing this element functioned as an androgen-specific enhancer in transiently transfected LNCaP-1F5 cells. ARE-SARG+4.6 cooperated with flanking sequences for optimal activity. Inactivation of ARE-SARG+4.6 completely abolished the androgen response of the enhancer. Chromatin immunoprecipitation (ChIP) experiments showed chromatin structural changes of the enhancer in the presence of R1881. ARE-SARG+4.6 was able to bind to the androgen receptor, but not to the glucocorticoid receptor, correlating with its androgen-specific activity in transfections.
Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development
Authors: Quignodon L , Legrand C , Allioli N , Guadaño-Ferraz A , Bernal J , Samarut J , Flamant F
Published: December 2004

We have generated transgenic reporter mice to analyze the spatio-temporal distribution of thyroid hormone signaling during mouse brain development. The reporter system, utilizing a chimeric yeast Gal4 DNA-binding domain–thyroid hormone α ligand-binding domain fusion protein to drive lacZ expression, revealed that thyroid hormone signaling starts in the midbrain roof several days before the onset of thyroid gland function, and that it remains highly heterogeneous in the central nervous system throughout pre- and postnatal development. We speculate that this heterogeneity might provide neural cells with positional information during development.
Duplicated receptors for VIP and PACAP (VPAC1R and PAC1R) in a teleost fish, Fugu rubripes
Authors: Cardoso J C R , Power D M , Elgar G , Clark M S
Published: December 2004

Two principal groups of receptors orthologous with human PAC1R and VPAC1R and were identified and characterised at the genomic level in the teleost fish Fugu rubripes. An additional group orthologous with VPAC2R was also identified and partially characterised. In Fugu, gene duplication of each of the PAC1Rs, VPAC1Rs and VPAC2Rs appears to have occurred. The topology of the tree surrounding the Fugu duplications and other isolated piscine sequences indicates that the duplication events for these six genes clearly preceded the speciation event leading to the Cypriniformes and Tetraodontiformes and is probably teleost-specific. Overall, the combined pattern of gene expression for each pair of duplicated genes mirrored the expression in other vertebrates. However, within each pair of duplicates further specialisation had occurred, with each demonstrating differential tissue distribution profiles suggesting they that may be responsible for the divergent action of the ligands, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). The Fugu VPAC1R gene regions showed conserved synteny with human chromosome 3p21.3 and also C. elegans chromosome X, indicating that the putative ancestral human chromosome 3 region may be equivalent to chromosome X in Caenorhabditis elegans.
Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct
Authors: Davies J S , Holter J L , Knight D , Beaucourt S M , Murphy D , Carter D A , Wells T
Published: December 2004

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.
Cloning of glucocorticoid-regulated genes in C6/ST1 rat glioma phenotypic reversion
Authors: Valentini S R , Armelin M C S
Published: December 2004

The C6 rat glioma cell line is responsive to glucocorticoid hormones. C6 variants that are hyper-responsive (ST1) and resistant (P7) to hormone treatment have been derived previously. Glucocorticoid treatment of ST1 cells leads to complete reversion of the transformed phenotype and loss of tumorigenic potential. Production of C type retrovirus particles is also induced by glucocorticoids in ST1 cells. Cloning of the genes regulated by glucocorticoids in this cell system was used here as a strategy to uncover the gene products involved in the transformed-to-normal phenotypic change. Construction of a cDNA library from glucocorticoid-treated ST1 cells and screening by differential hybridization resulted in the isolation of three cellular sequences that code for rat metallothioneins (C27 and C41) and α1-acid glycoprotein (C36). Northern blot analysis revealed that expression of these genes was dramatically induced by hydrocortisone in ST1 but not in P7 cells. Viral genomic RNA was used to isolate and characterize retrovirus-related sequences that could also be responsible for the phenotypic reversion phenomenon.

Journal of Endocrinology (1996) 148, 11–17
Regional differences and up-regulation of progesterone receptors in adipose tissues from oestrogen-treated sheep
Authors: Mayes J S , McCann J P , Ownbey T C , Watson G H
Published: December 2004

Differing risk factors between men and women for a number of vascular and metabolic diseases have been linked to regional obesity. The differences in the distribution of adipose tissues between men (abdominal or upper-body obesity) and women (gluteal/femoral or lower body obesity) suggest a role for sex steroids in the regional distribution of fat. Previous work from this laboratory has shown the presence of oestrogen receptor (ER) in gluteal, perirenal and omental adipose tissues of ewes with similar physical characteristics to the ER in uterine tissue. The concentration profile for adipose ER was gluteal> perirenal>omental. In this report, we determined the physiological significance of adipose ERs by showing an up-regulation of the progesterone receptor (PR) in adipose tissues after oestrogen treatment in a fashion similar to that seen in a major responsive tissue such as uterus. Using PR antibodies (PR-6 and C-262), Western blot analysis of PR from oestrogen-treated sheep indicated that PR was induced in uterus>>>gluteal adipose>perirenal adipose consistent with the concentration of ER contained in these tissues. PR could not be detected by Western blotting in omental adipose tissue from oestrogen-treated animals or in gluteal, perirenal and omental adipose tissues from untreated animals. Sucrose gradient profiles of progestin (R-5020) binding from uterus and gluteal adipose tissues of oestrogen-treated ewes showed specific binding in both the 5S and 9S regions of the gradient, while perirenal and omental adipose tissue had only the 5S peak. The amount of specific binding was increased with oestrogen treatment in all the tissues. When gluteal adipose tissue cytosol was preincubated with PR antibody (C-262) to prevent binding of ligand and subjected to sucrose gradient analysis, both the 5S and 9S regions were diminished, suggesting that both peaks contained PR. Dilution of uterine cytosol resulted in an increase in the ratio of the 5S to the 9S peak, indicating that the 9S PR complex dissociates at low concentrations; this may be the reason why only the 5S peak was observed in perirenal and omental adipose tissues. These data offer further support for a direct role of sex steroids in regional adipose accretion and metabolism.

Journal of Endocrinology (1996) 148, 19–25
The proliferative responses of porcine thyroid follicular cells to epidermal growth factor and thyrotrophin reflect the autocrine production of transforming growth factor-β1
Authors: Cowin A J , Heaton E L , Cheshire S H , Bidey S P
Published: December 2004

The present study has investigated an involvement of autocrine transforming growth factor-β1 (TGF-β1) in regulating the proliferative response of porcine thyroid follicular cells (TFCs) to epidermal growth factor (EGF) and TSH. Primary monolayer TFC cultures exposed to EGF over the range 0–0·4 nmol/l showed a dose-dependent increase in [methyl-3H]thymidine incorporation, whereas higher EGF doses were associated with a reduction in the level of [methyl-3 H]thymidine incorporation. TGF-β immunoneutralisation had little effect on the stimulatory action of low EGF doses, but led to an increase in [methyl-3H]thymidine incorporation at higher EGF levels. In TFC cultures exposed to TSH, the level of [methyl-3H]thymidine incorporation attained at a dose of 1 U TSH/1 was enhanced in the presence of TGF-β1 antiserum, although the similar stimulatory effect of 8-bromo cAMP was unaffected. Treatment of TFCs with phorbol 12-myristate 13-acetate (8 nmol/l) to activate protein kinase C (PKC) led to an enhanced incorporation of [methyl-3H]thymidine which was increased further after neutralisation of endogenous TGF-β1. While confirming, therefore, a role for autocrine TGF-β1 in maintaining control of TFC DNA synthesis in vitro, these findings provide evidence that an increase in the availability of autocrine TGF-β1 effected by EGF and TSH may play an instrumental role in limiting the cellular hyperplasia induced by these factors within the thyroid follicular microenvironment. Moreover, the present data also suggest that the availability of active autocrine TGF-β1 to TFCs under such conditions may be dependent upon a PKC-mediated mechanism.

Journal of Endocrinology (1996) 148, 87–94
IGF-II and IGF-binding proteins increase dramatically during rabbit pregnancy
Authors: Nason K S , Binder N D , Labarta J I , Rosenfeld R G , Gargosky S E
Published: December 2004

During pregnancy, changes in the IGF axis are associated with changes in maternal metabolism and nutrient repartitioning which are necessary to meet the demands of a growing conceptus. The aim of this study was to assess the IGF axis, maternal weight changes and food intake in female New Zealand White rabbits (n=7) prior to breeding (day 0) and serially throughout pregnancy until term (day 30-31).

The total weight of the pregnant does progressively increased from 4·03±0·06 kg (mean ± s.e.m.) on day 0 to 4·47 ±0·07 kg on day 30 (P<0·001). Maternal tissue mass (total weight minus estimated conceptus weight) increased until day 18, plateaued to day 22/23, and then significantly declined. On day 30, the maternal tissue mass was not significantly different from the non-pregnant value, such that the final increase in total weight was due to conceptus growth. Although the does were fed ad libitum, food intake did not change until day 29 when it decreased to approximately 50% of previous intake (P<0·01).

Maternal serum IGF-I was 499 ± 32 ng/ml on day 0, reached a peak of 832 ± 160 ng/ml on day 21 (P<0·02), and then declined to 341 ± 49 ng/ml on day 30. In contrast, serum IGF-II increased dramatically from a non-pregnant level of 85 ±14 ng/ml to 16 295 ± 2488 ng/ml on day 23 (P<0·001), and then rapidly declined (3335 ± 954 ng/ml, day 30). Changes in serum IGF-binding proteins (IGFBPs) followed a pattern similar to IGF-II, as assessed by Western-ligand blotting. All IGFBPs, especially the 45–40 kDa IGFBP-3 doublet, increased dramatically between days 12 and 24 of pregnancy, and then declined towards term.

In conclusion, we observed unique and dramatic changes in the maternal serum IGF axis that corresponded to periods of maternal weight gain and loss. The tissue source of IGFs and IGFBPs remains undetermined, although it is of note that the time when major changes in the IGF axis were first observed coincided with the time of functional change from yolk sac to placenta in the rabbit.

Journal of Endocrinology (1996) 148, 121–130
Effects of hormone replacement therapy on platelet membrane fatty acid composition
Authors: Ranganath L R , Christofides J A , Wright J W , Marks V
Published: December 2004

Oestrogen replacement therapy has been shown to protect postmenopausal women from ischaemic heart disease, strokes and hypertension. The mechanism of protection conferred by oestrogen, although partly attributable to changes in serum lipoproteins, is not fully understood. The present study was undertaken to assess the effect of hormone replacement therapy on the composition of platelet membrane fatty acids in postmenopausal women. These were analysed by gas-liquid chromatography before and six weeks after continuous conjugated equine oestrogen therapy (0·625 mg daily) combined with cyclical therapy with 75 μg l-norgestrel from day 17 to 28 of a 28-day cycle. Each subject acted as her own control. The principal findings of the study were that, following treatment, there was a 16·2% reduction in platelet membrane polyunsaturated fatty acids (P<0·001), an increase of 9·1 and 7·1% in saturated fatty acids and monounsaturated fatty acids respectively (P<0·001) and a 17·8% reduction in arachidonic acid (P<0·003). There was no correlation between changes in membrane fatty acids and serum lipoproteins. This suggests that the changes in membrane composition noted in this study may be a primary effect of hormone replacement therapy, especially oestrogen.

Journal of Endocrinology (1996) 148, 207–212
Peripheral and ovarian IGF-I concentrations during the ovine oestrous cycle
Authors: Leeuwenberg B R , Hudson N L , Moore L G , Hurst P R , McNatty K P
Published: December 2004

IGF-I was measured by RIA in plasma samples collected 8-hourly for 24 days which included two consecutive preovulatory surges of LH. In a separate study, ovarian venous blood was collected from animals undergoing ovariectomy on day 10 of the oestrous cycle, or 36 h later after being treated with prostaglandin with or without steroid-free bovine follicular fluid. Jugular venous blood samples were collected before, during and after surgery. Follicles were dissected from ovaries of these animals and sorted into categories of small, intermediate and large, non-atretic or atretic, and the follicular fluid was pooled and assayed for IGF-I. From another population of ovaries recovered from the slaughterhouse, granulosa, theca and corpora lutea were isolated, homogenized and assayed for IGF-I. Finally ovarian corpora lutea and granulosa cells were each incubated with tritiated amino acids overnight at 37 °C. Thereafter the tissues and media were sonicated, IGF-I extracted from the supernatant and tritiated IGF-I precipitated using a specific IGF-I antibody.

The absence of any significant change in peripheral IGF-I concentrations following ovariectomy and the finding that the ovarian venous IGF-I concentrations (161 ± 10 μg/l) were not significantly different from levels seen in peripheral blood (157 ± 10 μg/l) indicated that the ovary is not a net exporter of IGF-I. However, the ovary does synthesize IGF-I, as evidenced by granulosa and luteal synthesis, but probably not in quantities in excess of that utilized by ovarian tissues per se. Although the plasma IGF-I levels increased around the second preovulatory LH surge, the results overall indicated that the IGF-I concentrations in plasma are not strictly related to any major ovarian event during the oestrous cycle in the sheep. This view is based on the findings that the concentration of IGF-I in follicular fluid was not related to follicular health but correlated with those in peripheral plasma and that the ovarian venous concentrations did not vary between left and right ovaries irrespective of whether the ovaries contained a corpus luteum, dominant follicle or neither. Collectively, these results are consistent with the notion that IGF-I of ovarian origin fulfils an autocrine/paracrine function and does not have an endocrine role. Moreover, the results show that the concentrations of IGF-I in follicular fluid reflect those in peripheral plasma.

Journal of Endocrinology (1996) 148, 281–289
Mediation by the central nervous system is critical to the in vivo activity of the GH secretagogue L-692,585
Authors: Hickey G J , Drisko J , Faidley T , Chang C , Anderson L L , Nicolich S , McGuire L , Rickes E , Krupa D , Feeney W , Friscino B , Cunningham P , Frazier E , Chen H , Laroque P , Smith R G
Published: December 2004

To investigate the effect of hypophyseal transection (HST) on GH secretagogue activity of the non-peptidyl GH secretagogue L-692,585 in the conscious pig, male castrated swine were randomly assigned to either a hypophyseal stalk transection group (HST; n=3) or to a sham-operated control group (SOC; n=3). Treatments administered were L-692,585 (100 γg/kg), human GH-releasing factor(1–29)NH2 (GRF; 20 γg/kg) or L-692,585 (100 γg/kg) + GRF (20 γg/kg) on days −7 to −3 before surgery and days +3 to +8 after surgery. To evaluate the integrity of the pituitary gland, the animals were challenged with corticotropin-releasing hormone (CRH; 150 γg) or GnRH (150 ng/kg) both before and after surgery. Blood was collected from −60 to +180 min post treatment and assayed for GH, cortisol and LH.

Before surgery, no significant difference (P>0·05) in peak GH response (ng/ml) was present between the two groups (SOC vs HST) in response to L-692,585 (101 ± 12 vs 71 ± 9) or L-692,585 + GRF (171 ± 21 vs 174 ± 21). Only two out of three SOC vs three out of three HST pigs responded to GRF (13 ± 2 vs 25 ± 3) resulting in a significant difference between groups. Following surgery, significant differences were present in peak GH response (ng/ml) between SOC and HST groups following L-692,585 (79 ± 6 vs 13·8 ± 1·0); however, the response to L-692,585 + GRF was similar (115 ± 8 vs 94 ± 7). All animals responded to GRF; however, a significant difference was present between groups due to the magnitude of the responses. Whereas the cortisol responses (ng/ml) to L-692,585 in the SOC and HST groups were similar before surgery, a significant difference was present after surgery (44·4 ± 6·4 vs 14·6 ± 2·1). No significant difference was noted between the HST and SOC groups in response to CRH or GnRH either before or after surgery.

These results indicated that L-692,585 induced an immediate GH response in the intact animal in contrast to GRF where the GH release was variable. L-692,585 also stimulated an immediate increase in cortisol levels. Transection of the hypophyseal stalk dramatically decreased but did not ablate the GH or cortisol response to L-692,585. Co-administration of L-692,585 + GRF induced an immediate GH response of similar magnitude in the intact and HST animal. We conclude that L-692,585 has a direct but limited action at the level of the pituitary and that an intact hypophyseal stalk is required for a maximal GH and cortisol response. L-692,585 acts with GRF at the level of the pituitary to induce a maximal GH response. These findings suggest that L-692,585 stimulates GH secretion by acting in combination with GRF and interrupting the inhibitory tone of somatostatin on the somatotroph.

Journal of Endocrinology (1996) 148, 371–380
Characteristics of BRIN-BG5 and BRIN-BG7, two novel glucose-responsive insulin-secreting cell lines produced by electrofusion
Authors: McClenaghan N H , Barnett C R , O'Harte F P M , Swanston-Flatt S K , Ah-Sing E , Flatt P R
Published: December 2004

Two hybrid insulin-secreting cell lines (BRIN-BG5 and BRIN-BG7) were established by the novel approach of electrofusing RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Cells were selected from the fusion mixture on the basis of insulin output. Wells showing five to ten times greater insulin output than parental RINm5F cells were selected, subcultured and cloned. Clonal BRIN-BG5 and BRIN-G7 cells grow as monolayers with epithelial morphology. The differences in doubling time of 28 and 20 h respectively were associated with morphological differences; the growth pattern and insulin content of each cell line remaining stable for over 50 passages. In acute 20-min tests, both cell lines showed peak secretory responses (1·9- and 1·8-fold respectively) to 8·4 mmol/l glucose. Membrane depolarization with 25 mmol/l K+ evoked 3·7- and 3·9-fold increases in insulin output. l-Alanine (10 mmol/l) also served to promote 2·4- and 1·6-fold increases in insulin release respectively. Increasing the Ca2+ concentration from 1·28 to 7·68 mmol/l potentiated this effect by 1·8- and 1·5-fold. Incubation with forskolin (25 μmol/l) or phorbol-12-myristate 13-acetate (10 nmol/l), in the presence of l-alanine, similarly enhanced the secretory effect on BRIN-BG5 and BRIN-BG7 cells by 1·3- to 2·1-fold and 1·2- to 1·5-fold respectively. The presence of a functional glucose-sensing mechanism in both cell lines was confirmed by the demonstration of the glucose transporter GLUT-2 and measurement of glucokinase activity. These functional properties suggest that insulin-secreting BRIN-BG5 and BRIN-BG7 cells represent two useful glucoseresponsive cell lines for future studies of the function of the pancreatic B-cell.

Journal of Endocrinology (1996) 148, 409–417
Follicular variation in ovarian 11β-hydroxysteroid dehydrogenase (11βHSD) activities: evidence for the paracrine inhibition of 11βHSD in human granulosa-lutein cells
Authors: Michael A E , Gregory L , Thaventhiran L , Antoniw J W , Cooke B A
Published: December 2004

We have previously shown that detectable metabolism of cortisol to cortisone by 11β-hydroxysteroid dehydrogenase (11βHSD) in human granulosa-lutein cells, pooled for each patient from all aspirated ovarian follicles, is associated with failure to conceive by in vitro fertilization and embryo transfer. The aims of the present study were to assess: (1) the variation in the 11β-HSD activities of granulosa-lutein cells obtained from individual follicles in relation to oocyte maturity and (2) whether the 11βHSD activity of pooled granulosa-lutein cells reflects the 11βHSD activities of the individual follicles for a given patient. 11βHSD activities were measured in intact cells in serum-free medium by a radiometric conversion assay (100 nmol/l [ 3H]cortisol to [3H]cortisone). Follicular 11βHSD activities ranged from <10 (undetectable) to 514 pmol/mg protein per 4 h (n=105 follicles from 12 patients) and did not correlate with oocyte maturity. In three separate patients, the follicular 11βHSD activities ranged from <10 to 117 pmol/mg protein per 4 h (n=8 follicles), 19 to 514 pmol/mg per 4 h (n=9) and 60 to 390 pmol/mg per 4 h (n=8). The 11βHSD activities of the corresponding multi-follicular pools of cells were <10, <10 and 44 pmol/mg per 4 h respectively, all of which were significantly lower (P<0·05) than the arithmetic means for the activities in the individual follicles (52, 132 and 215 pmol/mg per 4 h respectively). Likewise, the 11βHSD activities of two independent multi-patient pools of cells were significantly lower than the mean values of the 11βHSD activities of the appropriate individual patients. We conclude that ovarian 11βHSD activity varies between follicles and that co-culture of granulosa-lutein cells with low enzyme activity can suppress the ovarian 11βHSD activity in cells from different follicles (or patients) with high rates of cortisol metabolism. Hence, these data indicate the potential for paracrine inhibition of ovarian 11βHSD activity in human granulosa-lutein cells.

Journal of Endocrinology (1996) 148, 419–425
11β-Hydroxysteroid dehydrogenase in developing rat intestine
Authors: Pácha J , Mikšík I
Published: December 2004

The enzyme 11β-hydroxysteroid dehydrogenase (11β-OHSD) prevents the binding of corticosterone to mineralocorticoid receptors by reversible conversion of biologically active corticosterone to inactive 11-dehydrocorticosterone. To clarify the relationship between high plasma concentrations of corticosterone during weaning and high activity of intestinal transport pathways that are induced by aldosterone in immature intestine, we have studied the distribution, developmental pattern and regulation of 11 β-OHSD in intestinal segments that possess mineralocorticoid target epithelium. Dehydrogenase activity was already high in the caecum, and the proximal and distal colon on the second postnatal day and altered little until adulthood. In contrast, the activity in the ileum was low during the first two weeks of life, rose more than 5-fold in the next 20 days to attain a peak in 30-day-old rats, and thereafter declined to the values of adult animals. There was no significant reductase activity (conversion of 11-dehydrocorticosterone to corticosterone) in any intestinal segment of young and adult rats. The regulation of intestinal 11β-OHSD by corticosteroids and thyroid hormones was studied in the ileum and distal colon. In weanling rats, adrenalectomy or a high-salt diet decreased 11β-OHSD activities in both intestinal segments whereas dexamethasone administration prevented this decline in adrenalectomized rats and administration of deoxycorticosterone acetate led to a significant increase of intestinal 11β-OHSD activities in rats kept on a high-salt diet. Dexamethasone administration to intact adult rats also stimulated 11 β-OHSD activity in the ileum and distal colon. The changes in thyroid status of weanling rats did not change the 11β-OHSD activities. We conclude that (1) the developmental patterns of 11β-OHSD activity in the small and large intestine are not identical and this discrepancy may facilitate the maturation effect of glucocorticoids in the small intestine and the stimulatory effect of aldosterone in the large intestine and (2) corticosteroids but not thyroid hormones can modulate 11β-OHSD activity in the developing intestine.

Journal of Endocrinology (1996) 148, 561–566
Characterization of transforming growth factor-α receptors in the avian ovary: alterations in ligand binding to granulosa cells during follicular maturation
Authors: Onagbesan O M , Woolveridge I , Peddie M J
Published: December 2004

The presence of epidermal growth factor receptors (EGF-R) and the ligands epidermal growth factor/transforming growth factor-α (EGF/TGFα) have been reported in mammalian ovaries where they are implicated in folliculogenesis and steroidogenesis. Evidence is presented to show that authentic EGF/TGFα receptors are expressed by the avian granulosa cells. The TGFα receptors (TGFα-R) from chicken granulosa cells were characterized by specific binding of 125I-human TGFα. In this study, competition with human EGF, human TGFα, human IGF-I, human basic fibroblast growth factor (bFGF) and insulin for 125I-human TGFα binding demonstrated that the avian granulosa cell TGFα-R binds human EGF with 300-fold lower affinity than human TGFα. IGF-I, bFGF and insulin did not displace bound 125I-TGFα. Scatchard analysis showed that a single class of high-affinity binding sites is present on the granulosa cells (Kd 0·23 ± 0·009 nm). However, the number of binding sites altered during follicular maturation with a significant decline in the most mature follicle. These results go some way to explaining the basis for the changing sensitivity of avian granulosa cells to EGF/TGFα stimulation as they mature. In addition, the gonadotrophins, LH and FSH, increased the number of receptors in cultured granulosa cells and may therefore partially influence folliculogenesis and steroidogenesis through this route.

Journal of Endocrinology (1996) 149, 171–179
Influence of nutrition on hepatic IGF-I mRNA levels and plasma concentrations of IGF-I and IGF-II in meat-type chickens
Authors: Kita K , Tomas F M , Owens P C , Knowles S E , Forbes B E , Upton Z , Hughes R , Ballard F J
Published: December 2004

We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards.

In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and β-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to β-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations.

Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition.

IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment.

Journal of Endocrinology (1996) 149, 181–190
Expression of IGF-I and IGF-binding protein genes in cirrhotic liver
Authors: Ross R J M , Chew S L , D'Souza Li L , Yateman M , Rodriguez-Arnao J , Gimson A , Holly J , Camacho-Hubner C
Published: December 2004

The liver plays a central role in the IGF-I axis producing the majority of circulating hormone and some of its binding proteins (IGFBPs). Cirrhosis of the liver is characterised by changes in IGF-I and IGFBPs associated with liver fibrosis and regeneration. We have studied steady state levels of mRNA for the genes in the IGF-I axis in normal and cirrhotic human liver, localised the most highly expressed gene, IGFBP-1, and measured circulating IGFBP-3 by radioimmunoassay (RIA), IGFBP-2 and IGFBP-3 by Western ligand blot (WLB), and protease activity for IGFBP-3 in cirrhotic patients. Messenger RNA for IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 was detectable by Northern blotting in normal and cirrhotic liver although there was considerable variation in expression. IGFBP-2 and IGFBP-3 tended to be more highly expressed in cirrhotic liver and IGFBP-1 was more highly expressed in normal liver, although there were no significant differences. In normal liver, in situ hybridisation localised IGFBP-1 to hepatocytes. In cirrhotic liver the regenerating nodules showed expression of IGFBP-1 while there was none in fibrotic tissue. Circulating IGFBP-3 levels were low as measured by RIA and WLB but protease activity was only found in one patient. IGFBP-2 levels, assessed by WLB, were similar to the normal serum pool. Our data show that key mRNAs involved in the IGF-I axis continue to be expressed in cirrhotic liver despite end stage liver disease. The low levels of IGFBP-3 do not appear to be due to reduced gene transcription or increased protease activity.

Journal of Endocrinology (1996) 149, 209–216