bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluctuating Estrogen and Progesterone Receptor Expression in Brainstem Norepinephrine Neurons through the Rat Estrous Cycle*
Authors: Haywood Suzy A. , Simonian Sharon X. , van der Beek Eline M. , Bicknell R. John , Herbison Allan E.
Published: January 2014

Norepinephrine (NE) neurons within the nucleus tractus solitarii (NTS; A2 neurons) and ventrolateral medulla (A1 neurons) represent gonadal steroid-dependent components of several neural networks regulating reproduction. Previous studies have shown that both A1 and A2 neurons express estrogen receptors (ERs). Using double labeling immunocytochemistry we report here that substantial numbers of NE neurons located within the NTS express progesterone receptor (PR) immunoreactivity, whereas few PRs are found in ventrolateral medulla. The evaluation of ERα and PR immunoreactivity in NE neurons through the estrous cycle revealed a fluctuating pattern of expression for both receptors within the NTS. The percentage of A2 neurons expressing PR immunoreactivity was low on metestrus and diestrus (3–7%), but increased significantly to approximately 24% on proestrous morning and remained at intermediate levels until estrus. The pattern of ERα immunoreactivity in A2 neurons was more variable, but a similar increment from 11% to 40% of NE neurons expressing ERα was found from diestrus to proestrus. Experiments in ovariectomized, estrogen-treated and estrogen-plus progesterone-treated rats revealed that PR immunoreactivity in A2 neurons was induced strongly by estrogen treatment, whereas progesterone had no significant effect. The numbers of ERα-positive NE neurons were not influenced by steroid treatment. These observations provide direct evidence for PRs in NE neurons of the brainstem and show that cyclical patterns of gonadal steroid receptor expression exist in A2, but not A1, neurons through the rat estrous cycle. The expression of PR in A2 neurons appears to be driven principally by circulating estrogen concentrations. The fluctuating levels of ERα and PR expression in these brainstem NE neurons may help generate cyclical patterns of biosynthetic and electrical activity within reproductive neural networks.
Recombinant E-Peptides of Pro-IGF-I Have Mitogenic Activity
Authors: Tian X. C. , Chen Maria J. , Pantschenko Alex G. , Yang T. J. , Chen Thomas T.
Published: January 2014

In mammals, the post-translational proteolytic modification of many pro-hormones generates multiple peptides with similar or distinct biological activities. The production of mature insulin-like growth factor I (IGF-I) involves the cleavage of an E-peptide from pro-IGF-I. Although the IGF-I E-peptides are conserved among vertebrate species, their fate and biological roles have not been identified. To test whether the E-peptides possess any biological activity, three recombinant E-peptides of pro-IGF-I, namely Ea-2-, Ea-3- and Ea-4-peptides of rainbow trout, Oncorhynchus mykiss, were produced in vitro with a His-tag expression system and partially purified with an affinity Ni++ column. The mitogenic activity of each E-peptide was determined by (1) the stimulation of total DNA content increase as measured by a fluorometric method and/or (2) stimulation of [3H]-thymidine incorporation into DNA. Recombinant Ea-4-peptide elicited a dose-related increase in both mitogenic assays in NIH3T3 cells. To further test the specificity of the mitogenic activity of Ea-4-peptide, three other cell lines were used: retroviral transformed human embryonic kidney cells (293GP), human mammary gland tumor cells (MCF-7) and caprine mammary epithelial cells (CMEC). Similar levels of mitogenic activity were observed in all cell lines tested for Ea-4-peptide. Mitogenic activity was also observed with recombinant Ea-2- and Ea-3-peptides when assayed in NIH3T3 cells. These results suggest that E-peptides of rainbow trout pro-IGF-I possess mitogenic activity in heterologous systems.
Transcriptional Regulation of Human 11β-Hydroxylase (hCYP11B1)
Authors: Wang Xiao-Li , Bassett Mary , Zhang Yin , Yin Su , Clyne Colin , White Perrin C. , Rainey William E.
Published: January 2014

Steroid 11β-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11β-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5′-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing −1102 bp of hCYP11B1 5′-flanking DNA (pB1–1102). An element at position −71/−64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. In addition, Western analysis of H295R and adrenal lysates demonstrated expression of high levels of ATF-2 and ATF-1. CRE-binding protein levels varied among the strains of H295R cells tested. Transcription of CYP11B1 also appeared to be regulated by steroidogenic factor-1 (SF-1). Luciferase reporter gene activity was increased after cotransfection with expression vectors containing SF-1. An element in hCYP11B1 at positions −242/−234 (CCAAGGCTC), previously termed Ad4, was required for maximal induction by SF-1 and was found to bind SF-1 in electrophoretic mobility shift assays. The key role for SF-1 in hCYP11B1 transcription is in contrast to its lack of an effect on expression of the hCYP11B2 (aldosterone synthase) isozyme. The differential effects of SF-1 on transcription of hCYP11B1 and hCYP11B2 may be one of the mechanisms controlling differential expression of these isozymes within the zonae fasciculata and glomerulosa of the human adrenal cortex.
Dynamic Regulation of Mouse Ovarian Stanniocalcin Expression during Gestation and Lactation*
Authors: Deol Harminder K. , Varghese Robin , Wagner Graham F. , DiMattia Gabriel E.
Published: January 2014

Stanniocalcin is a glycoprotein hormone that appears to play a paracine/autocrine role in several mammalian tissues. Recently studies have shown that stanniocalcin is highly expressed in the ovaries of mice and humans and we have investigated its expression in the mouse ovary during several physiological states to identify potential functional relationships. During postnatal development the pattern of stanniocalcin (STC) gene expression begins to become thecal-restricted as early as day 5 and achieves the adult pattern of expression by two weeks of age. During postnatal development the primary sites of STC protein localization are the theca and oocytes and after maturation it is also strongly concentrated in the corpora lutea. Over the estrous cycle the pattern of both STC gene expression and protein localization do not show dramatic changes though STC immunoreactivity (STCir) staining appears to be greatest during metestrus I. In the superovulation model, however, we observed a significant increase in STC messenger RNA (mRNA) levels after treatment with hCG implying regulation by LH. During gestation the expression of ovarian STC increases 15-fold and is localized to the theca-interstitial cells with lower expression also being found in the corpora lutea. STC also becomes detectable in the serum for the first time suggesting an endocrine role for STC during gestation. Interestingly, the presence of a nursing litter appears to up-regulate STC gene expression in lactating mice suggesting a role for ovarian STC in lactation. Also striking is the intense STCir staining found in oocytes as they are devoid of STC mRNA, thus implying a role for STC in oocyte maturation. Stanniocalcin, to our knowledge, is unique because no other secreted proteins produced by the ovarian thecal-interstitial compartment are significantly induced during mouse pregnancy. In summary, our data provide evidence for the active regulation of STC expression in the ovary during gestation and lactation and therefore implies that STC is a new regulator of the gestational and nursing state.
Glucocorticoids Regulate Plasma Membrane Potential During Rat Thymocyte Apoptosis in Vivo and in Vitro
Authors: Mann Cynthia L. , Cidlowski John A.
Published: January 2014

Glucocorticoids induce a series of profound biochemical changes in thymocytes that initiate apoptosis; however, the pathways beyond receptor transactivation that lead to this form of cell death are not fully understood. In this study, we report a novel site of action for glucocorticoids at the site of the plasma membrane. Specifically, we find that glucocorticoids induce the loss of plasma membrane potential both in vivo and in vitro. The glucocorticoid-induced loss of plasma membrane potential in cultured primary isolated rat thymocytes was both dose and time dependent. Other steroid hormones, including progesterone, estrogen, and testosterone, fail to alter the depolarization state of the thymocyte plasma membrane. Interestingly, other nonsteroid stimuli that also activate apoptosis in thymocytes also lead to cellular depolarization. In contrast, HeLa cells, which contain functional glucocorticoid receptors but do not die in response to hormone, do not alter their plasma membrane potential in response to glucocorticoids, indicating a strong association between depolarization and apoptosis. Furthermore, the ability of glucocorticoids to depolarize the plasma membrane of thymocytes required the interaction of glucocorticoids with their cognate receptor, because RU486 failed to depolarize thymocytes and antagonized the effect of glucocorticoids. Finally, experiments using inhibitors of transcription and translation indicated that the loss of plasma membrane potential in thymocytes following glucocorticoid treatment required de novo gene expression. The results of these studies establish that the loss of plasma membrane potential is an early important feature of glucocorticoid-induced apoptosis of thymocytes.
Prostaglandin E2 (EP1) Receptor Agonist-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: The Involvement of TGF-α
Authors: Kimura Mitsutoshi , Osumi Sachie , Ogihara Masahiko
Published: January 2014

We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10−6m), PGE2 (10−6m), and 17-phenyl-trinor-PGE2 (10−9m) in a time- and dose-dependent manner. PGE2 was less potent than 17-phenyl-trinor-PGE2 in stimulating hepatocyte mitogenesis. Sulprostone (10−6m) and 11-deoxy-PGE1 (10−6m) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE2, 17-phenyl-trinor-PGE2, and sulprostone were abolished by treatment with a specific EP1 receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP1 receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked by specific inhibitors of growth-related signal transducers, such as genistein, wortmannin, PD98059, and rapamycin. A monoclonal antibody against TGF-α dose-dependently inhibited PGE2- and 17-phenyl-trinor-PGE2-induced hepatocyte mitogenesis. Treatment with the EP1 receptor agonists significantly increased the secretion of TGF-α, reaching a maximum within 5 min. The increase in TGF-α secretion was blocked by SC-51322, U-73122, somatostatin, and verapamil and potentiated by ionomycin. These results indicate that the proliferative mechanisms of action of EP1 receptor agonists are mediated through an increase in the autocrine secretion of TGF-α, which is dependent on the EP1 receptor/G-protein involved in PLC regulation/PLC/Ca2+ system. The locally secreted TGF-α, in turn, acts as a complete mitogen that stimulates the tyrosine kinase/MAPK pathway in these cells.
Grtp1, A Novel Gene Regulated by Growth Hormone
Authors: Lu Chunxia , Kasik John , Stephan Dietrich A. , Yang Shaohua , Sperling Mark A. , Menon Ram K.
Published: January 2014

An in vitro model of GH-responsive cells was subjected to microarray analysis to identify a novel gene regulated by GH. This 258 amino acid protein, we term GH Regulated TBC Protein-1 (GRTP1), contains the TBC signature motif of GTPase activator proteins of Rab-like small GTPases. Northern blot analysis revealed a 1.3 kb major mRNA species, most abundant in testes. TaqMan assay confirmed that in the mouse, Grtp1 is expressed at highest levels in testes, with lesser abundance in intestine, kidney, lung, and liver. In the testis, expression of Grtp1 significantly increases post-pubertally. Administration of GH to mice increased levels of GRTP1 mRNA in testes (140%), but decreased GRTP1 mRNA abundance in kidney (50%) and liver (25%). Grtp1 was localized to mouse proximal chromosome 8. Orthologs of this protein are present in human, mouse, rat, and drosophila suggesting that GRTP1 has an important biological role(s).
In Vivo Gonadotropin-Releasing Hormone Secretion in Female Rats during Peripubertal Development and on Proestrus*
Authors: Sisk Cheryl L. , Richardson Heather N. , Chappell Patrick E. , Levine Jon E.
Published: January 2014

Pubertal development in female rats is characterized by increased LH levels and the appearance of estrogen-dependent afternoon LH mini-surges. In these studies we performed the first analysis of GnRH patterns in peripubertal rats to determine whether there are similar changes in pulsatile GnRH release. Microdialysis samples were collected at 5-min intervals throughout a 5-h afternoon period from 22 rats sampled on a single day between 30–47 days of age. Adult female rats were sampled on proestrus for comparison. In 30- to 33-day-old rats, GnRH release was infrequent (2.7 pulses/5 h; n = 3), whereas intermediate pulse frequencies were observed in 34- to 37-day-old rats (6.4 pulses/5 h; n = 9) and 38- to 42-day-old (5.0 pulses/5 h; n = 5) rats. The highest GnRH pulse frequencies were observed in 43- to 47-day-old rats (9.4 pulses/5 h; n = 5). Mean GnRH pulse amplitude did not vary significantly with age. Animals sampled before vaginal opening (VO) exhibited significantly slower GnRH pulse frequencies than those sampled after vaginal opening (1.3 pulses/5 h pre-VO vs. 7.6 pulses/5 h post-VO; P= 0.01). An afternoon increase in GnRH secretion, defined operationally as a greater than 25% increase in mean GnRH levels in the last half of the sampling period and tentatively termed a mini-surge, was observed in 0%, 33%, 40%, and 60% of 30- to 33-, 34- to 37-, 38- to 42-, and 43- to 47-day-old rats, respectively. An overall increase in GnRH pulse frequency was observed in females displaying a mini-surge (9.0 pulses/5 h with mini-surge compared with 4.7 pulses/5 h with no mini-surge). The mini-surge itself, however, was associated with a late afternoon increase in GnRH pulse amplitude and not in pulse frequency. In adult proestrous rats, peak levels during the GnRH surge were an order of magnitude greater than those reached in pubertal animals. Our findings demonstrate that pubertal maturation in the female rat is associated with an acceleration of GnRH pulse generator activity and that later stages of pubertal maturation are characterized by the appearance of afternoon increases in GnRH release that may underlie previously reported mini-surges in LH.
Activation of cAMP-Dependent Protein Kinase Increases the Protein Level of Steroidogenic Factor-1
Authors: Æsøy Reidun , Mellgren Gunnar , Morohashi Ken-Ichirou , Lund Johan
Published: January 2014

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is an essential regulator of endocrine organogenesis, sexual differentiation, and steroidogenisis. SF-1 is a transcriptional regulator of cAMP responsive genes, but the exact mechanisms by which cAMP-dependent PKA modulates SF-1 dependent transcription leading to increased steroidogenic output have not been determined. In this report the effects of PKA activation on SF-1 in living cells have been examined by the use of full-length SF-1 cDNA fused to the cDNA encoding green fluorescent protein (GFP). The GFP-SF-1 fusion protein localized to the nucleus of both steroidogenic Y1 cells and nonsteroidogenic COS-1 cells, and the functional properties of wild-type SF-1 were conserved. When the catalytic subunit of PKA was coexpressed with GFP-SF-1, we observed that the fluorescence emission was markedly elevated. These findings were confirmed by Western blot analysis, showing that stimulation of PKA increased SF-1 protein levels. The PKA- induced expression of SF-1 protein was not accompanied by an increase in SF-1 mRNA levels. However, pulse-chase studies showed a decrease in SF-1 degradation rate in response to activation of PKA, indicating that PKA elevates the level of SF-1 by increasing the stability of SF-1 protein.
Intensive Direct Cavernous Sinus Sampling Identifies High-Frequency, Nearly Random Patterns of FSH Secretion in Ovariectomized Ewes: Combined Appraisal by RIA and Bioassay
Authors: Clarke Iain , Moore Lloyd , Veldhuis Johannes
Published: January 2014

Analyses of FSH secretion suggest pulsatile, nonpulsatile, or compositely pulsatile and nonpulsatile release modes. This may reflect the reduced signal-to-noise ratio inherent in FSH pulse estimation procedures and/or immunological-biological assay inconsistencies. To address these issues, we sampled cavernous sinus and jugular venous blood concomitantly from ovariectomized sheep at either 5-min or 1-min intervals. Samples from the former were assayed by RIA, and those from the latter by RIA and bioassay. Waveform-independent peak detection revealed FSH pulses occurring at high frequency. Pulsatile FSH secretion accounted for 28% of total secretion. Approximate entropy analysis showed that FSH secretion was nearly random. There was synchronous release of LH and FSH, but most FSH secretion was not associated with LH release; 13% of discrete FSH and LH pulses were concordant. We infer that FSH secretion exhibits pulsatile and basal/nonpulsatile features, with high-entropy features. Linear and nonlinear statistical measures revealed joint sample-by-sample synchrony of FSH and LH release, indicating pattern coordination despite sparse synchrony of pulses. We postulate that pattern synchrony of FSH and LH release is effected at the level of the gonadotrope. Concordant FSH and LH pulses probably result from pulsatile GnRH input, but other mechanisms could account for independent FSH pulses.
Proliferation of Rhesus Ovarian Surface Epithelial Cells in Culture: Lack of Mitogenic Response to Steroid or Gonadotropic Hormones
Authors: Wright Jay W. , Toth-Fejel SuEllen , Stouffer Richard L. , Rodland Karin D.
Published: January 2014

Ovarian cancer is the most lethal gynecological cancer, and approximately 90% of ovarian cancers derive from the ovarian surface epithelium (OSE), yet the biology of the OSE is poorly understood. Factors associated with increased risk of nonhereditary ovarian cancer include the formation of inclusion cysts, effects of reproductive hormones and the number of ovulations experienced in a woman’s lifetime. Distinguishing between these factors is difficult in vivo, but cultured OSE cells are viable tools for some avenues of research. Here we establish rhesus macaque OSE cultures and demonstrate that these cells express cytokeratin, vimentin, N-cadherin, ER-α, and PR but are negative for E-cadherin. We show that these cells activate MAPK and proliferate in response to extracellular calcium, as do human and rat OSE. In contrast, the gonadotropic hormones FSH (4–400 IU/liter), LH (8.5–850 IU/liter), and human CG (10–1000 IU/liter) fail to stimulate proliferation. We find that concentrations of progesterone and estrogen normally present in follicles just before ovulation (∼1000 ng/ml) significantly decrease the number of mitotically active rhesus macaque OSE cells as determined by PCNA labeling, total cell count, and 3H-thymidine uptake, whereas lower steroid concentrations have no effect.
Gestational and Lactational Exposure of Male Mice to Diethylstilbestrol Causes Long-Term Effects on the Testis, Sperm Fertilizing Ability in Vitro, and Testicular Gene Expression
Authors: Fielden Mark R. , Halgren Robert G. , Fong Cora J. , Staub Christophe , Johnson Larry , Chou Karen , Zacharewski Tim R.
Published: January 2014

The objective of the study was to determine the long-term effects of gestational and lactational exposure to diethylstilbestrol (DES; 0, 0.1, 1, and 10 μg/kg maternal body weight) on mouse testicular growth, epididymal sperm count, in vitro fertilizing ability, and testicular gene expression using cDNA microarrays and real-time PCR in mice on postnatal day (PND) 21, 105, and 315. In the high dose group there was a persistent decrease in the number of Sertoli cells, and sperm count was decreased on PND315 (P < 0.05). Sperm motion was unaffected; however, the in vitro fertilizing ability of epididymal sperm was decreased in the high dose group on both PND105 (P < 0.001) and PND315 (P < 0.05). Early and latent alterations in the expression of genes involved in estrogen signaling (estrogen receptor α), steroidogenesis (steroidogenic factor 1, 17α-hydroxylase/C17,20-lyase, P450 side chain cleavage, steroidogenic acute regulatory protein, and scavenger receptor class B1), lysosomal function (LGP85 and prosaposin), and regulation of testicular development (testicular receptor 2, inhibin/activin β C, and Hoxa10) were confirmed by real-time PCR. The results demonstrate that early exposure to DES causes long-term adverse effects on testicular development and sperm function, and these effects are associated with changes in testicular gene expression, even long after the cessation of DES exposure.
Vitamin D3 Is More Potent Than Vitamin D2 in Humans
Authors: Heaney Robert P. , Recker Robert R. , Grote James , Horst Ronald L. , Armas Laura A. G.
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism, published December 22, 2010, 10.1210/jc.2010-2230
Allelic Deletion in Pituitary Adenomas Reflects Aggressive Biological Activity and Has Potential Value as a Prognostic Marker1
Authors: Bates A. S. , Farrell W. E. , Bicknell E. J. , McNicol A. M. , Talbot A. J. , Broome J. C. , Perrett C. W. , Thakker R. V. , Clayton R. N.
Published: January 2014

Tumors of the pituitary gland are usually benign adenomas and account for 10% of all intracranial neoplasms. Five pituitary tumors have previously been reported to harbor multiple allelic deletions. Of these, three displayed particularly aggressive biological behavior, whereas there were no clinical details provided for the others. This study was designed to test the hypothesis that genetic deletions are a marker of invasive behavior and to identify the loci most commonly involved. Accordingly, we studied two cohorts of pituitary tumors, classified radiologically as invasive or noninvasive, for loss of heterozygosity (LOH).

There is a significantly higher frequency of LOH in invasive tumors (10.8% of all loci examined) compared to noninvasive tumors (2.4%; P < 0.001). Of the 11 loci investigated, 75% of the allelic deletions identified in invasive tumors were found at 4 loci: 11q13, 13q12–14, 10q, and 1p. Twenty of 47 invasive tumors had evidence of at least 1 allelic deletion, whereas 14 of 20 had more than 1. Of the 6 tumors with only 1 deletion, 5 involved the 11q13 locus, suggesting that this is an early change in the transition from noninvasive to invasive adenoma. Comparison of invasive and noninvasive tumors demonstrates a significantly higher frequency of deletions affecting 11q13 (P < 0.001), 13q12–14 (P < 0.05), and 10q26 (P < 0.05) in invasive tumors. In addition, allelic deletion correlates with increasingly invasive behavior (modified Hardy classification), as 73% of grade 4 tumors compared to 33% of grade 3 and 9.5% of grade 1 and 2 tumors demonstrated LOH at any locus. Furthermore, in some tumors we identified a breakpoint between markers intragenic and extragenic to the retinoblastoma gene (Rb1) on chromosome 13q, suggesting that tumor suppressor genes other than or in addition to Rb1 may be involved in pituitary tumorigenesis. This was further supported by the presence of Rb protein in two of four tumors where the genetic loss extended to include the intragenic marker D13S153.

Early identification of tumors with likely invasive potential by means of genetic analysis (LOH) may provide useful information on potential tumor behavior and aid tumor management in a manner that is not possible using routine histological methods. A large prospective study is required in patients without radiological evidence of invasion to assess the value of LOH in predicting outcome and for planning treatment.
Clonal Origin of Toxic Thyroid Nodules with Constitutively Activating Thyrotropin Receptor Mutations
Authors: Krohn Knut , Führer Dagmar , Holzapfel Hans-Peter , Paschke Ralf , Führer-Sakel Dagmar
Published: January 2014

Constitutively activating TSH receptor mutations have recently been detected in toxic nodules. In vitro studies suggest that mutated receptor signaling constitutively elevates cAMP, which causes hyperfunction and proliferation of thyrocytes. Therefore, toxic nodules with constitutively activating somatic TSH receptor mutations should result from clonal expansion of a single mutated cell. To test this hypothesis, we studied the clonal origin of 27 toxic nodules. In 13 of 27 nodules, a somatic mutation in the TSH receptor was identified. A PCR-based clonality assay that analyzes X-chromosome inactivation was used. The assay amplifies a polymorphism located in the androgen receptor gene. Of 27 toxic nodules studied, 23 (85%) were informative for the polymorphism. In the group that contains a somatic mutation in the TSH receptor, 10 of 11 cases showed nonrandom X inactivation, indicating clonal expansion. In only one toxic nodule with a TSH receptor mutation was random X inactivation detected. In the group without detectable mutations in exons 9 and 10 of the TSH receptor and exons 7–10 of the Gsα protein, only 6 of 12 toxic nodules show nonrandom X-chromosome inactivation. Therefore, the majority of toxic nodules with constitutively activating TSH receptor mutations are of clonal origin. This finding supports the hypothesis that toxic nodules arise from aberrant growth of a single cell. It is widely accepted that somatic mutations might initiate monoclonal growth. The TSH receptor mutations in these toxic nodules together with Gsα mutations in others are the most likely candidates for the initiation of this thyroid tumor. The clonal origin of toxic nodules in the group without detected mutations in the TSH receptor or the Gsα protein suggests somatic mutations in genes that are unknown to date.
Interferon-γ and Activin A Promote Insulin-Like Growth Factor-Binding Protein-2 and -4 Accumulation by Human Luteinizing Granulosa Cells, and Interferon-γ Promotes Their Apoptosis1
Authors: Cataldo Nicholas A. , Fujimoto Victor Y. , Jaffe Robert B.
Published: January 2014

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) antagonize IGF and gonadotropin actions on granulosa cells. Human atretic follicles express IGFBP-2 in granulosa cells more strongly and contain higher levels of IGFBP-2 and IGFBP-4 than healthy follicles. We studied the effects of interferon-γ (IFNγ) and activin A, which decrease progesterone accumulation, on granulosa cell IGFBP production and apoptosis. Conditioned media from luteinizing granulosa cells cultured with IFNγ or activin A and/or LH were subjected to ligand blotting; northern blots of total ribonucleic acid (RNA) from these cells were probed for IGFBP-2 and -4. Apoptosis was measured by in situ DNA end labeling. LH decreased medium IGFBP-2 to 21% of the control value. Although IFNγ did not alter basal medium IGFBP-2, in the presence of LH it increased IGFBP-2 3.4-fold, with parallel changes in messenger RNA levels. Activin A also tended to increase medium IGFBP-2 in LH-treated cultures. In conditioned medium, IGFBP-4 was consistently decreased by LH, whereas both IFNγ and activin A increased IGFBP-4 and decreased IGFBP-4 protease activity. Both LH and IFNγ modestly stimulated IGFBP-4 messenger RNA levels. Follistatin antagonized the action of activin A, but not that of IFNγ. IFNγ, but not activin A, increased granulosa cell apoptosis. In conclusion, IFNγ produced by activated lymphocytes may decrease endogenous IGF activity through stimulation of IGFBPs and may promote apoptosis of granulosa-lutein cells in vivo and, thus, luteal regression. Activin A similarly promotes IGFBP accumulation, but it does not promote apoptosis. (J Clin Endocrinol Metab 83: 179–186, 1998)
Longitudinal Reproductive Hormone Profiles in Infants: Peak of Inhibin B Levels in Infant Boys Exceeds Levels in Adult Men1
Authors: Andersson Anna-Maria , Toppari Jorma , Haavisto Anne-Maarit , Petersen Jørgen H. , Simell Tuula , Simell Olli , Skakkebæk Niels E.
Published: January 2014

The gonads are usually considered quiescent organs in infancy and childhood. However, during the first few postnatal months of life, levels of gonadotropins and sex hormones are elevated in humans. Recent epidemiological evidence suggests that environmental factors operating perinatally may influence male reproductive health in adulthood. The early postnatal activity of the Sertoli cell, a testicular cell type that is supposed to play a major role in sperm production in adulthood is largely unknown. Recently, the peptide hormone inhibin B was shown to be a marker of Sertoli cell function in the adult male. In the adult woman, inhibin B is secreted by the granulosa cells. Longitudinal serum levels of inhibin B were measured in healthy boys (n = 15) and girls (n = 15), in cord blood, and every third month during the first 2 yr of life. In addition, serum levels of FSH, LH, and testosterone (boys) were measured in the same group of children. In boys, inhibin B, FSH, LH, and testosterone levels were all elevated at 3 months of age. However, the peak of inhibin B was unexpectedly high, into the supraadult range (mean ± se, 378 ± 23 pg/mL) and persisted much longer than the elevation of FSH, LH, and testosterone. Thus, although levels of FSH, LH, and testosterone decreased into the range observed later in childhood by the age of 6–9 months, serum inhibin B levels remained elevated up to at least the age of 15 months. In girls, the hormonal pattern was generally more complex, with a high interindividual variation in levels of inhibin B, FSH, and LH within each age. In conclusion, the sustained elevation of inhibin B to supraadult levels in infant boys indicates that the neonatal period may be a developmental window important for Sertoli cell proliferation and maturation. Thus, the gonads may be potentially vulnerable to exogenous endocrine interference, e.g. from environmental factors during this period of life. Measurement of serum levels of inhibin B in infants may give clinical clues about developmental deficiencies in the gonads that otherwise only become apparent around puberty or later in life.
Age-Related Reduction in Glucose Elimination Is Accompanied by Reduced Glucose Effectiveness and Increased Hepatic Insulin Extraction in Man1
Authors: Ahrén Bo , Pacini Giovanni
Published: January 2014

This study examined whether insulin secretion, insulin sensitivity, glucose effectiveness (SG), and hepatic extraction (HE) of insulin are altered by age when glucose tolerance is normal. A frequently sampled iv glucose tolerance test was performed in 20 elderly (E, 10/10 male/female, all 63 yr old) and in 20 young subjects (Y, 10/10 male/female, all 27 yr old), who were similar in body mass index and 2-h blood glucose during oral glucose tolerance test. E exhibited impaired glucose elimination (iv tolerance index, 1.31 ± 0.10 vs. 1.70 ± 0.12% min−1; P = 0.019). First-phase insulin secretion and SI did not differ between the groups, whereas E had lower glucose sensitivity of second-phase insulin secretion (0.40 ± 0.07 vs. 0.70 ± 0.08 (pmol/L)min−2/(mmol/L), P = 0.026), lower SG, 0.017 ± 0.002 vs. 0.025± 0.002 min−1, P = 0.004), and higher HE (81.3 ± 2.4 vs. 73.2 ± 2.1%, P = 0.013). Across both groups, SG correlated positively with glucose tolerance index (r = 0.58, P < 0.001) and negatively with HE (r =− 0.54, P < 0.001). Plasma leptin and glucagon did not change by age, whereas plasma pancreatic polypeptide (PP) was higher in E (122 ± 18 vs.66 ± 6 pg/mL, P = 0.004). PP did not, however, correlate to any other parameter. We conclude that E subjects with normal oral glucose tolerance have reduced SG, impaired second-phase insulin secretion, and increased HE, whereas SI and first-phase insulin secretion seem normal. SG seems most related to age-dependent impairment of glucose elimination, whereas leptin, glucagon, and PP do not seem to contribute.
Direct Postoperative and Follow-Up Results of Transsphenoidal Surgery in 19 Acromegalic Patients Pretreated with Octreotide Compared to Those in Untreated Matched Controls
Authors: Biermasz N. R. , van Dulken H. , Roelfsema F.
Published: January 2014

In this study 19 patients were preoperatively treated with octreotide for 1–17 months (mean, 5 months), with doses from 150–1500 μg daily, and those patients were matched to 19 untreated patients with comparable tumor classification and preoperative serum GH concentrations. Octreotide was started at 300 μg daily by sc injections or continuous sc infusion using a pump in increasing doses, depending on the responses of the serum GH and insulin-like growth factor I (IGF-I) concentrations. During pretreatment, seven patients achieved a serum GH concentration below 5 mU/L, whereas six patients normalized their serum IGF-I.

Postoperatively, a serum GH concentration below 5 mU/L was achieved in 15 pretreated and 14 untreated patients, a normal serum IGF-I level (<2 sd) was achieved in 10 pretreated and 15 untreated patients, and normal serum GH suppression during GTT was reached in 12 treated and 14 control patients. No differences were found in complication rate or incidence of hypopituitarism caused by surgery.

Adjuvant therapy was required in 7 treated and 5 untreated patients. At follow-up examination, 5.7 and 4 yr postoperatively, 10 pretreated and 12 control patients could be considered cured by surgery only, according to our criteria for remission (serum GH, <5 mU/L; normal GH suppression and normal serum IGF-Ι). In summary, we found no difference in direct postoperative and follow-up results of transsphenoidal surgery between pretreated patients and untreated patients. This finding is in discordance with other studies, which have claimed a beneficial effect of octreotide pretreatment.
Role of Nutritional Status in the Regulation of Adrenarche1
Authors: Remer Thomas , Manz Friedrich
Published: January 2014

The factors regulating adrenarche are unknown. Recent in vitro studies have demonstrated that insulin and insulin-like growth factor I induce major adrenal steroidogenic enzyme genes and increase the production of adrenal androgens. Literature findings strongly suggest that changes in body mass index (BMI) reflect an integrated nonhormonal index of changes in serum levels and/or bioactivities of insulin and insulin-like growth factor I. We therefore longitudinally investigated individual changes in BMI and urinary 24-h excretion rates of dehydroepiandrosterone sulfate (DHEAS) in a prepuberty (PreC; n = 22, 11 boys and 11 girls) and a puberty (PubC; n = 20, 10 boys and 10 girls) cohort of healthy children. Twenty-four-hour urine samples were collected at yearly intervals during observation periods that lasted at least 4 yr (comprising ≥5 consecutive 24-h urine collections). For 4-yr intervals highly significant tracking coefficients (P < 0.001) of 0.73 (PreC) and 0.93 (PubC) were observed for DHEAS, emphasizing the importance of individual (and genetic) influences on adrenal androgen excretion. In both cohorts almost 3-fold higher median increases in urinary DHEAS excretion rates (P < 0.05) were observed during the 1-yr period of the individually highest rises in BMI compared with the 1-yr period of significantly lower rises in BMI (P < 0.01) in the same children after the factor age was controlled for. However, no consistently significant associations were found between urinary DHEAS output and BMI from simple cross-sectional correlations at defined age points. These findings provide the first in vivo evidence that a change in the nutritional status, measurable in the form of Δ-BMI (but not BMI alone), is an important physiological regulator of adrenarche regardless of individual adrenal androgen excretion level, age, and developmental stage.
Differentiation of Human Orbital Preadipocyte Fibroblasts Induces Expression of Functional Thyrotropin Receptor1
Authors: Valyasevi Rosanee W. , Erickson Dana Z. , Harteneck Debra A. , Dutton Charyl M. , Heufelder Armin E. , Jyonouchi Soma C. , Bahn Rebecca S.
Published: January 2014

Although the autoantigen involved in Graves’ hyperthyroidism is known to be the TSH receptor (TSHr), whether this antigen plays a primary role in the pathogenesis of Graves’ ophthalmopathy (GO) is unclear. We sought to determine whether fibroblasts derived from orbital adipose/connective tissue are capable of differentiating into adipocytes that bear immunoreactive and functional TSHr. In addition, we assessed relative levels of TSHr gene expression in normal and GO orbital adipose/connective tissue specimens.

GO and normal orbital preadipocyte fibroblasts, cultured under conditions known to stimulate adipocyte differentiation, showed evidence of adipogenesis and positive immunostaining for TSHr protein. In addition, significantly more cAMP was produced in response to TSH stimulation in the differentiated cultures than in undifferentiated cultures derived from the same individuals’ cells. Other studies demonstrated relatively greater TSHr gene expression in GO than in normal orbital tissue specimens.

These results indicate that orbital preadipocyte fibroblasts increase their TSHr expression with differentiation and suggest that these cells play an important role in the pathogenesis of GO. Furthermore, our studies support the concept that TSHr may be an important target antigen in this condition. Factors that stimulate adipocyte differentiation and TSHr expression in the orbit in GO have yet to be defined.
Glucocorticoid Replacement Is Permissive for Rapid Eye Movement Sleep and Sleep Consolidation in Patients with Adrenal Insufficiency1
Authors: GarcÍa-Borreguero Diego , Wehr Thomas A. , Larrosa Oscar , Granizo Juan J. , Hardwick Donna , Chrousos George P. , Friedman Theodore C.
Published: January 2014

There is a well described temporal relation between hormonal secretion and sleep phase, with hormones of the hypothalamic-pituitary-adrenal (HPA) axis possibly playing a role in determining entry into and duration of different sleep stages. In this study sleep features were studied in primary Addison’s patients with undetectable levels of cortisol treated in a double blind, randomized, cross-over fashion with either hydrocortisone or placebo supplementation. We found that REM latency was significantly decreased in Addison’s patients when receiving hydrocortisone at bedtime, whereas REM sleep time was increased. There was a trend toward an increase in the percentage of time in REM sleep and the number of REM sleep episodes. Waking time after sleep onset was increased, whereas no differences were observed between the two conditions when total sleep time or specific non-REM sleep parameters were evaluated. Our results suggest that in Addison’s patients, cortisol plays a positive, permissive role in REM sleep regulation and may help to consolidate sleep. These effects may be mediated either directly by the central effects of glucocorticoids and/or indirectly through CRH and/or ACTH.
Somatostatin Receptor Subtypes in Human Thymoma and Inhibition of Cell Proliferation by Octreotide in Vitro
Authors: Ferone Diego , van Hagen Martin P. , Kwekkeboom Dik J. , van Koetsveld Peter M. , Mooy Diana M. , Lichtenauer-Kaligis Elgin , Schönbrunn Agnes , Colao Annamaria , Lamberts Steven W. J. , Hofland Leo J.
Published: January 2014

Somatostatin (SS) and SS receptor (SSR) subtypes, code-named sst1–5, are heterogeneously expressed in the normal human thymus. This suggests their involvement in controlling the immune and/or neuroendocrine functions in this organ. Moreover, recently a high in vivo uptake of[ 111In-DTPA-d-Phe1]octreotide has been reported in patients bearing thymoma. The present study characterizes in vivo and in vitro, functional SS-binding sites in a human thymoma.

A high uptake of[ 111In-DTPA-d-Phe1]octreotide was observed in the chest of a patient with myasthenia gravis due to a cortical thymoma. Specific binding of[ 125I-Tyr11]SS-14 was found on a membrane preparation of the surgically removed thymoma. Scatchard analysis showed high affinity binding sites (Kd, 47.5 ± 2.5 pmol/L) with low maximum binding capacity (23.5 ± 2.5 fmol/mg membrane protein). RT-PCR analysis showed the presence of sst1, sst2A, and a predominant sst3 messenger RNA (mRNA) expression in the tumor tissue. Primary cultured tumor cells expressed sst3 mRNA only. In contrast to the normal thymus, SS mRNA was not expressed. By immunohistochemistry, the tumor cells highly expressed sst3 receptors, weakly expressed sst1 receptors, and showed no immunostaining for sst2A receptors. sst2A immunoreactivity was found in the stromal compartment of the tumor, particularly on the endothelium of small intratumoral blood vessels. In primary cultured tumor cells, both SS and octreotide (10 nmol/L) significantly inhibited[ 3H]thymidine incorporation by 40.6% and 43.2%, respectively.

The following conclusions were reached. 1) As this tumor displayed a high immunoreactivity for sst3 and the cultured tumor cells expressed the sst3 mRNA only, this SSR may be the subtype involved in the inhibition of epithelial tumor cell proliferation by octreotide in vitro. 2) A loss of endogenous SS production in this thymoma might be implicated in the uncontrolled cell growth. 3) In this case, the sst3 may play a role in determining the uptake of[ 111In-DTPA-d-Phe1]octreotide by in vivo SS receptor scintigraphy.
Preeclampsia Is Associated with Low Circulating Levels of Insulin-Like Growth Factor I and 1,25-Dihydroxyvitamin D in Maternal and Umbilical Cord Compartments*
Authors: Halhali Ali , Tovar Armando R. , Torres Nimbe , Bourges Hector , Garabedian Michele , Larrea Fernando
Published: January 2014

Insulin-like growth factor I (IGF-I) stimulates renal and placental 1,25-dihydroxyvitamin D [1,25-(OH)2D] and is considered an important regulator of fetal growth. As 1,25-(OH)2D and birth weight are low in preeclampsia, this study was undertaken to determine whether circulating levels of IGF-I were associated with serum 1,25-(OH)2D concentrations in preeclamptic (PE group) and normotensive (NT group) pregnancies. Maternal and umbilical cord serum levels of IGF-I and 1,25-(OH)2D were significantly (P < 0.01) lower in the PE group than in the NT group. The concentrations of these two hormones correlated significantly in the umbilical cord (P < 0.05) and in the maternal (P < 0.001) compartments of the PE and NT groups, respectively. The amount of IGFBP-3 was 64% lower whereas that of IGFBP-1 was 2.9-fold higher in umbilical cord serum of the PE group compared with the NT group. In addition, maternal and umbilical cord serum IGF-I correlated significantly (P < 0.05) with weight and length at birth only in the PE group. In conclusion, the results of this study indicate that circulating IGF-I and 1,25-(OH)2D levels in both maternal and umbilical cord compartments are low in preeclampsia. Furthermore, this study suggests a differential regulatory effect of IGF-I on 1,25-(OH)2D synthesis and fetal growth depending on the presence or absence of preeclampsia.
Continuation of Growth Hormone (GH) Replacement in GH-Deficient Patients during Transition from Childhood to Adulthood: A Two-Year Placebo-Controlled Study
Authors: Vahl Nina , Juul Anders , Jørgensen Jens O. L. , Ørskov Hans , Skakkebæk Niels E. , Christiansen Jens. S.
Published: January 2014

Previous studies have demonstrated beneficial effects of GH replacement, in adults with GH deficiency (GHD), on body composition, physical fitness, and quality of life. These studies, however, concern patients with adult-onset GHD or childhood-onset (CO) patients enrolled several years after withdrawal of initial therapy. So far, the effects of continuation of GH-administration in patients with CO-GHD have not been examined. We studied a group of nineteen young adults (13 males+ 6 females; 16–26 yr old; mean age, 20.2 ± 0.65 yr) with CO-GHD, in a randomized, parallel, double-blind, placebo-controlled trial for 1 yr, followed by an open phase with GH for 1 yr. All patients received GH therapy at the start of study, and trial medication (GH/placebo) was given in a similar dose. Patients randomized to continued GH treatment exhibited no significant changes in any parameters tested, but intra- and interindividual variations in insulin-like growth factor (IGF)-I levels could suggest compliance problems. Discontinuation of GH for 1 yr resulted in a decrease in serum IGF-I, from 422.0 ± 56.8 to 147.8 ± 33.4 μg/L, in the placebo group (P = 0.003). After discontinuation of GH for 1 yr, an increase in total body fat (TBF, kg), measured by dual-energy x-ray absorptiometry scan, was seen[ placebo: 22.7 ± 2.7 to 26.5 ± 2.5 (P = 0.01); GH:16.2 ± 2.1 to 17.2 ± 2.1 (not significant)]. Resumption of GH after placebo was followed by increments in serum IGF-I (μg/L) [from 147.8 ± 33.4 to 452 ± 76 (P = 0.001)] and IGF-binding protein 3, as well as in fasting glucose (mmol/L) [4.9 ± 0.2 vs. 5.3 ± 0.2 (P = 0.03)]. After resumption of GH lean body mass (kg) increased [52.4 ± 4.9 vs. 60.7 ± 5.6 (P = 0.006)]. Likewise, resumption of GH therapy increased thigh muscle volume and thigh muscle/fat ratio, as assessed by computed tomography [muscle volume (cm2/10 mm): 118.2 ± 11.7 vs. 130.0 ± 10.9 (P = 0.002); muscle/fat ratio: 1.33 ± 0.24 vs. 1.69 ± 0.36 (P = 0.02)].

In conclusion, discontinuation of GH treatment in GHD patients, during the transition from childhood to adulthood, induces significant and potentially unfavorable changes in IGF-I and body composition, both of which are reversed after resumption of GH treatment. By contrast, continuation of GH therapy results in unaltered IGF-I and body composition. We recommend continuation of GH therapy in these patients, to be undertaken in collaboration between pediatricians and adult endocrinologists.