bluuebunny/crossref_metadata_2025_split huggingface dataset:
Corticotropin-Releasing Factor (CRF) Agonists Stimulate Testosterone Production in Mouse Leydig Cells through CRF Receptor-1*
Authors: Heinrich Nadja , Meyer Mike R. , Furkert Jens , Sasse Annette , Beyermann Michael , Bönigk Wolfgang , Berger Hartmut 
Published: January 2014

AbstractThe influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nm for hCRF, antagonized by the CRF antagonist α-helical CRF9–41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to[ 11-d-Thr,12-d-Phe]- and[ 13-d-His,14-d-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2α or -R2β. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.
Expression of Growth Differentiation Factor-9 Messenger Ribonucleic Acid in Ovarian and Nonovarian Rodent and Human Tissues*
Authors: Fitzpatrick Susan L. , Sindoni Deborah M. , Shughrue Paul J. , Lane Malcolm V. , Merchenthaler Istvan J. , Frail Donald E. 
Published: January 2014

AbstractGrowth differentiation factor-9 (GDF-9) is a member of the transforming growth factor-β family that is reported to be expressed exclusively in the ovary, specifically in the oocyte. Female mice deficient in GDF-9 are infertile, suggesting that GDF-9 receptor agonists and antagonists may specifically modulate fertility. We now report that GDF-9 messenger RNA (mRNA) is expressed in nonovarian tissues in mice, rats, and humans. GDF-9 mRNA was detected in mouse and rat ovary, testis, and hypothalamus by Northern blot and RT-PCR analyses. The localization of GDF-9 mRNA specifically in oocytes of the mouse ovary was confirmed by in situ hybridization histochemistry. In mouse testis, although localization in Sertoli cell cytoplasm could not be ruled out, mRNA expression was observed in large pachytene spermatocytes and round spermatids. The expression of GDF-9 mRNA in human tissues was assessed by Northern blot and RT-PCR analyses. GDF-9 mRNA was observed in ovary and testis and, surprisingly, in diverse nongonadal tissues, including pituitary, uterus, and bone marrow. Therefore, GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Furthermore, human GDF-9 mRNA is expressed not only in the gonads, but also in several extragonadal tissues. The function and relevance of nongonadal GDF-9 mRNA are not known, but may affect strategies for contraception and fertility that are based on GDF-9 activity.
Regional Expression of Transforming Growth Factor-α in Rat Ventral Prostate during Postnatal Development, after Androgen Ablation, and after Androgen Replacement*
Authors: Banerjee Subhadra , Banerjee Partha P. , Zirkin Barry R. , Brown Terry R. 
Published: January 2014

AbstractThe prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-α (TGFα), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFα in the rat ventral prostate during postnatal development to adulthood. TGFα-immunopositive cells were located throughout the ductal epithelium from postnatal days 5–20. By day 45 and thereafter, regional variation in TGFα expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFα in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFα staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFα in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFα expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFα in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFα is not regulated by androgen in this region of the ventral prostate. In summary, TGFα expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFα may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.
Adrenal Steroid Regulation of Neurotrophic Factor Expression in the Rat Hippocampus
Authors: Chao Helen M. , Sakai Randall R. , Ma Li Yun , McEwen Bruce S. 
Published: January 2014

AbstractAdrenal steroids and neurotrophic factors are important modulators of neuronal plasticity, function, and survival in the rat hippocampus. Adrenal steroids act through two receptor subtypes, the glucocorticoid receptor (GR) and the mineralocorticoid receptor, and activation of each receptor subtype has distinct biochemical and physiological consequences. Adrenal steroids may exert their effects on neuronal structure and function through the regulation of expression of neurotrophic and growth-associated factors. We have examined adrenal steroid regulation of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and basic fibroblast growth factor, as well as the growth associated protein GAP-43, through activation of GR or mineralocorticoid receptor with selective agonists. Our findings indicated that in CA2 pyramidal cells, adrenalectomy resulted in decreases in the levels of basic fibroblast growth factor and neurotrophin-3 messenger RNA, which were prevented by activation of mineralocorticoid but not glucocorticoid receptors. Adrenalectomy-induced increases in GAP-43 and brain-derived neurotrophic factor messenger RNA levels could be blocked by activation of glucocorticoid receptors in CA1, but not in CA3, pyramidal cells. Thus the extent to which adrenal steroids regulate hippocampal neurotrophic and growth-associated factors, appears to be dependent both on the adrenal steroid receptor subtype activated and on the hippocampal subregion examined.
Prolonged Depletion of Guanosine Triphosphate Induces Death of Insulin-Secreting Cells by Apoptosis*
Authors: Li Guodong , Segu Venkatesh Babu G. , Rabaglia Mary E. , Luo Rui-Hua , Kowluru Anjaneyulu , Metz Stewart A. 
Published: January 2014

AbstractInhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting β-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by[ 3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 μg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 μg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10–100μ m for 2–3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces β-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the β-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.
In Vitro Characterization of Somatostatin Receptors in the Human Thymus and Effects of Somatostatin and Octreotide on Cultured Thymic Epithelial Cells
Authors: Ferone Diego , van Hagen P. Martin , van Koetsveld Peter M. , Zuijderwijk Joke , Mooy Diana M. , Lichtenauer-Kaligis Elgin G. R. , Colao Annamaria , Bogers Ad J. J. C. , Lombardi Gaetano , Lamberts Steven W. J. , Hofland Leo J. 
Published: January 2014

AbstractSomatostatin (SS) and its analogs exert inhibitory effects on secretive and proliferative processes of various cells via high affinity SS receptors (SS-R). SS analogs bind with different affinity to the five cloned SS-R subtypes. Octreotide, an octapeptide SS analog, binds with high affinity to the SS-R subtype 2 (sst2). SS-R have been demonstrated in vivo and in vitro on cells from endocrine and immune systems. Among the lymphatic tissues, the thymus has been shown to contain the highest amount of SS, suggesting a local functional role of the peptide.We investigated the SS distribution and SS-R expression pattern in the normal human thymus using autoradiography, membrane homogenate binding studies, and RT-PCR. In addition, the effect of SS and octreotide on growth of cultured thymic epithelial cells (TEC) was studied.By autoradiography, binding of[ 125I-Tyr0]-SS-28 and[ 125I-Tyr3]-octreotide was detected in all seven thymuses studied. Specific[ 125I-Tyr3]-octreotide binding was shown on membrane preparations from thymuses, while not from cultured thymocytes. RT-PCR showed the expression of sst1, sst2A and sst3 messenger RNA (mRNA) in the thymic tissue, whereas sst1 and sst2A mRNAs were found in isolated TEC. SS mRNA was present in thymic tissue and in isolated TEC. SS and octreotide significantly inhibited 3H-thymidine incorporation in 3 of 3 and 6 of 6 TEC cultures, respectively. The percent inhibition ranged from 38.8 to 66.8% for SS and from 19.1 to 59.5% for octreotide.In conclusion, SS mRNA and sst1, sst2A, and sst3 mRNAs are expressed in the normal human thymus. Cultured TEC selectively express sst1 and sst2A mRNA and respond in vitro to SS and octreotide administration with an inhibition of cell proliferation. These data suggest a paracrine/autocrine role of SS and its receptors in the regulation of cell growth in thymic microenvironment.
Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture**This work was supported by consecutive program grants (Regkey 943208 and 983212) from the National Health and Medical Research Council of Australia.
Authors: Hertan Ruth , Farnworth Paul Graham , Fitzsimmons Karen Lesley , Robertson David Mark 
Published: January 2014

AbstractThe aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 m HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62–0.77 and an iodine incorporation ratio of 1.7–2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nm and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (&lt;2%), transforming growth factor-β (&lt;0.2%), or follistatin (≪0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.
Androgen Receptor, Estrogen Receptor α, and Estrogen Receptorβ Show Distinct Patterns of Expression in Forebrain Song Control Nuclei of European Starlings1
Authors: Bernard Daniel J. , Bentley George E. , Balthazart Jacques , Turek Fred W. , Ball Gregory F. 
Published: January 2014

Abstract
In songbirds, singing behavior is controlled by a discrete network of interconnected brain nuclei known collectively as the song control system. Both the development of this system and the expression of singing behavior in adulthood are strongly influenced by sex steroid hormones. Although both androgenic and estrogenic steroids have effects, androgen receptors (AR) are more abundantly and widely expressed in song nuclei than are estrogen receptors (ERα). The recent cloning of a second form of the estrogen receptor in mammals, ERβ, raises the possibility that a second receptor subtype is present in songbirds and that estrogenic effects in the song system may be mediated via ERβ. We therefore cloned the ERβ complementary DNA (cDNA) from a European starling preoptic area-hypothalamic cDNA library and used in situ hybridization histochemistry to examine its expression in forebrain song nuclei, relative to the expression of AR and ERα messenger RNA (mRNA), in the adjacent brain sections. The starling ERβ cDNA has an open reading frame of 1662-bp, predicted to encode a protein of 554 amino acids. This protein shares greater than 70% sequence identity with ERβ in other species. We report that starling ERβ is expressed in a variety of tissues, including brain, pituitary, skeletal muscle, liver, adrenal, kidney, intestine, and ovary. Similar to reports in other songbird species, we detected AR mRNA-containing cells in several song control nuclei, including the high vocal center (HVc), the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum. We detected ERα expression in the medial portion of HVc (also called paraHVc) and along the medial border of the caudal neostriatum. ERβ was not expressed in HVc, in the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, in the robust nucleus of the archistriatum, or in area X. In contrast, ERβ mRNA-containing cells were detected in the caudomedial neostriatum and medial preoptic area in a pattern reminiscent of P450 aromatase expression in the same brain regions in other songbirds. These data suggest that estrogenic effects on the song system are not mediated via ERβ-producing cells within song nuclei. Nonetheless, the overlapping expression of ERβ- and aromatase-producing cells in the caudomedial neostriatum suggests that locally synthesized estrogens may act via ERβ, in addition to ERα, to mediate seasonal or developmental effects on nearby song nuclei (e.g. HVc).
Down-Regulation of Protein Kinase C Inhibits Insulin-Like Growth Factor I-Induced Vascular Smooth Muscle Cell Proliferation, Migration, and Gene Expression1
Authors: Yano Kenji , Bauchat Jeanette R. , Liimatta Marya B. , Clemmons David R. , Duan Cunming 
Published: January 2014

AbstractInsulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, directed migration, differentiation, and apoptosis. The signaling mechanisms used by IGF-I to elicit these actions, however, are not well defined. In this study, we examined the role(s) of protein kinase C (PKC) in mediating the IGF-I actions in cultured porcine VSMCs. Out of the eleven known members of PKC family, PKC-α, -βI, -ε, -η, -λ,θ , and -ζ, were detectable by Western immunoblot analysis in these cells. Further analysis indicated that the subcellular distribution of several PKC isoforms is regulated by IGF-I. While IGF-I stimulated membrane translocation of PKC-η, -ε, and -ζ and regulated the cytosolic levels of PKC-βI, it had no such effect on PKC-α and -λ. To examine whether PKC activation is required for the IGF-I-regulated biological responses, phorbol myristate acetate (PMA) and GF109203X were used to down-regulate or inhibit PKC activity. Both PMA (1 μm) and GF109203X (20 μm) nearly completely suppressed the total PKC activity after a 30-min incubation (&gt; 90%), and this inhibition lasted for at least 24 h. Down-regulation or inhibition of PKC activity abolished the IGF-I-induced DNA synthesis, migration and IGFBP-5 gene expression. In contrast, the IGFBP-5 expression induced by forskolin was unaffected by PKC down-regulation or inhibition, suggesting that PKC activation is required for the IGF-regulated but not the cAMP-regulated events. Because the actions of IGF-I on DNA synthesis and IGFBP-5 gene expression in VSMCs have been shown to be mediated through the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway in porcine VSMCs, the potential role of PKC in IGF-I-induced activation of PI3 kinase and PKB/Akt were examined. Treatment with either PMA or GF109203X did not significantly affect the effects of IGF-I on PI3 kinase activation or PKB/Akt phosphorylation. These results indicated that PKC-βI, -η, -ε, and -ζ may play an essential role(s) in IGF-I regulation of VSMC migration, DNA synthesis and gene expression, and that these PKC isoforms may either act independently of the PI3 kinase pathway or act further downstream of PKB/Akt in the IGF signaling network.
Adrenal Capillary Endothelial Cells Stimulate Aldosterone Release through a Protein That Is Distinct from Endothelin1
Authors: Rosolowsky Lori J. , Hanke Craig J. , Campbell William B. 
Published: January 2014

AbstractWe tested the possibility that bovine adrenal capillary endothelial cells (ECs) stimulate aldosterone secretion from bovine zona glomerulosa (ZG) cells by the release of a transferable factor. In coincubations of ZG cells and ECs in serum-free medium, aldosterone release was stimulated approximately 17-fold, and the stimulation was related to the concentration of ECs. The maximal stimulation by ECs was 75% of the maximal response to ACTH. In contrast, adrenal pericytes and fibroblasts were without effect. ECs incubated alone without ZG cells did not produce aldosterone. Conditioned medium from ECs (EC-CM), but not adrenal fibroblasts, stimulated aldosterone release approximately 3-fold. The stimulation increased with the concentration of EC-CM and the duration of conditioning time. Steroidogenic activity in EC-CM was abolished by pronase treatment, indicating that the active factor was a protein. However, the activity in EC-CM was distinct from that of endothelin-1 (ET-1), an endothelial peptide that also stimulates aldosterone secretion, as it was not blocked by the ETB receptor antagonist PD-145065, it did not alter[ 125I]ET-1 binding to ZG cells, and its release occurred before the release of ET-1. Neither ECs nor EC-CM stimulated the production of cortisol from zona fasciculata cells. The activity of EC-CM was not blocked by an angiotensin II AT1 receptor antagonist or a bradykinin B2 receptor antagonist. EC-CM stimulated increased intracellular calcium in fura-2-loaded ZG cells, but did not increase the production of cAMP. Using gel filtration, this peptide had an approximate molecular mass of 3000 Da and migrated earlier than ET-1. This study demonstrates that ECs in vitro alter steroidogenesis through the release of a transferable substance and suggests the existence of an endothelium-derived steroidogenic factor that is produced by adrenal capillary ECs. This endothelium-derived steroidogenic factor may function in the adrenal gland as a paracrine regulator of aldosterone secretion.