bluuebunny/crossref_metadata_2025 huggingface dataset:
Saturated FFAs, Palmitic Acid and Stearic Acid, Induce Apoptosis in Human Granulosa Cells
Authors: Mu Yi-Ming , Yanase Toshihiko , Nishi Yoshihiro , Tanaka Atsushi , Saito Masayuki , Jin Cheng-Hao , Mukasa Chizu , Okabe Taijiro , Nomura Masatoshi , Goto Kiminobu , Nawata Hajime
Published: January 2014

Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.
Steroid-Independent Activation of ER by GnRH in Gonadotrope Pituitary Cells
Authors: Demay F. , De Monti M. , Tiffoche C. , Vaillant C. , Thieulant M. L.
Published: January 2014

In the rat pituitary gland the mechanism responsible for ERα regulation has not been fully elucidated. Using transient transfection assays in αT3–1 cells, a cell line of gonadotrope origin, we show that GnRH stimulates estrogen response element-containing promoters in an estrogen-independent manner. This effect was strictly ER and GnRH receptor dependent, as no activation of the reporter gene was observed in presence of the anti-estrogen ICI 182,780 or a GnRH antagonist. These data suggest that the GnRH-triggered signaling pathway results in 17β-estradiol-independent trans-activation of the ERα in αT3–1 cells. Furthermore, an additive activation was achieved when cells were treated with both GnRH and 17β-estradiol. In primary pituitary cells, GnRH alone (100 nm) did not cause a significant stimulation of reporter gene activity, presumingly due to the low amount of gonadotropes. Interestingly, the combination of 17β-estradiol and GnRH resulted in a significant increase in ERα trans-activation compared with that in cells treated with 17β-estradiol alone. This enhancement was prevented by ICI 182,780, showing an ERα requirement. Moreover, we show that the effects of GnRH on ERα transcriptional activity in gonadotrope cell lines are mediated by the PKC/MAPK pathway. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of ERα trans-activation in gonadotrope cells.
DNA Methylation Regulates Placental Lactogen I Gene Expression
Authors: Cho Jae-Hyeon , Kimura Hiromichi , Minami Tatsuya , Ohgane Jun , Hattori Naka , Tanaka Satoshi , Shiota Kunio
Published: January 2014

Expression of rat placental lactogen I is specific to the placenta and never expressed in other tissues. To obtain insight into the mechanism of tissue-specific gene expression, we investigated the methylation status in 3.4 kb of the 5′-flanking region of the rat placental lactogen I gene. We found that the distal promoter region of the rat placental lactogen I gene had more potent promoter activity than that of the proximal area alone, which contains several possible cis-elements. Although there are only 17 CpGs in the promoter region, in vitro methylation of the reporter constructs caused severe suppression of reporter activity, and CpG sites in the placenta were more hypomethylated than other tissues. Coexpression of methyl-CpG-binding protein with reporter constructs elicited further suppression of the reporter activity, whereas treatment with trichostatin A, an inhibitor of histone deacetylase, reversed the suppression caused by methylation. Furthermore, treatment of rat placental lactogen I nonexpressing BRL cells with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation, or trichostatin A resulted in the de novo expression of rat placental lactogen I. These results provide evidence that change in DNA methylation is the fundamental mechanism regulating the tissue-specific expression of the rat placental lactogen I gene.
Evidence That the Caudal Brainstem Is a Target for the Inhibitory Effect of Leptin on Food Intake
Authors: Grill Harvey J. , Schwartz Michael W. , Kaplan Joel M. , Foxhall James S. , Breininger John , Baskin Denis G.
Published: January 2014

Three experiments were performed to investigate the hypothesis that leptin action within the caudal brain stem (CBS) contributes to its intake inhibitory effects. The first experiment evaluated the anatomical distribution of leptin receptor mRNA in rat CBS using a sensitive fluorescence in situ hybridization method with a riboprobe specific for the long form of the leptin receptor (Ob-Rb). An Ob-Rb mRNA hybridization signal was detected in neurons of several CBS nuclei involved in the control of food intake, including the dorsal vagal complex and parabrachial nucleus. A strong hybridization signal was also obtained from neuronal cell bodies of a number of other structures including the hypoglossal, trigeminal, lateral reticular, and cochlear nuclei; locus ceruleus; and inferior olive. The anatomical profile revealed by fluorescence in situ hybridization was in good agreement with immunocytochemical analysis with an antibody specific to Ob-Rb. In a second experiment, exploring the relevance of CBS Ob-Rb to feeding behavior, rats were given a fourth intracerebroventricular (i.c.v.) injection of leptin (0.1, 0.83, or 5.0μ g; n = 9–11/group) or vehicle 30 min before lights-out on three consecutive days The two higher doses reduced food intake significantly at 2, 4, and 24 h after injection and caused significant reductions of body weight. The dose-response profiles for fourth i.c.v. administration were indistinguishable from those obtained from separate groups of rats that received leptin via a lateral i.c.v. cannula. In the last experiment, a ventricle-subthreshold dose of leptin (0.1 μg) microinjected unilaterally into the dorsal vagal complex suppressed food intake at 2, 4, and 24 h. The results indicate that the CBS contains neurons that are potentially direct targets for the action of leptin in the control of energy homeostasis.
Estradiol (E2) Enhances Neurite Outgrowth by Repressing Glial Fibrillary Acidic Protein Expression and Reorganizing Laminin
Authors: Rozovsky Irina , Wei Min , Stone David J. , Zanjani Hadi , Anderson Christopher P. , Morgan Todd E. , Finch Caleb E.
Published: January 2014

Neuronal remodeling in response to deafferenting lesions in the brain can be enhanced by estradiol (E2). Astrocytes are among the targets of E2 in complex interactions with neurons and may support or inhibit neuronal remodeling. In ovariectomized female rats given entorhinal cortex lesions, E2 replacement inhibited the increase of glial fibrillary acidic protein (GFAP) protein. To model the role of E2 in these complex processes, we used the “wounding-in-a-dish” of astrocyte-neuron cocultures. Low physiological E2 (1 pm) blocks the wound-induced increase of GFAP expression (transcription and protein) and enhances neurite outgrowth. The transcriptional responses to E2 during wounding are mediated by sequences in the 5′-upstream region of the rat GFAP promoter. Concurrently, E2 reorganized astrocytic laminin into extracellular fibrillar arrays, which others have shown support neurite outgrowth. The inhibition of GFAP expression by E2 in this model is consistent with in vivo findings that E2 enhanced recovery from deafferenting cortical lesions by increased neurite outgrowth in association with decreased GFAP expression. More generally, we hypothesize that physiological variations in E2 levels modulate neuronal plasticity through direct effects on GFAP transcription that, in turn, modify GFAP-containing intermediate filaments and reorganize astrocytic laminin.
Activation of Antimetastatic Nm23-H1 Gene Expression by Estrogen and Its α-Receptor
Authors: Lin Kwang-Huei , Wang Won-Jing , Wu Yi-Hsin , Cheng Sheue-Yann
Published: January 2014

Metastasis of various malignant cells is inversely related to the abundance of the Nm23-H1 protein. The role of estrogens in tumor metastasis has now been investigated by examining the effect of E2 on the expression of the Nm23-H1 gene. Three human breast carcinoma cell lines, in which endogenous ERα is expressed at different levels, were used as a tool to assess the role of ERα in Nm23-H1 gene-mediated metastasis. E2 induced time-dependent increases in the abundance of Nm23-H1 mRNA and protein, with the extent of these effects correlating with the level of expression of ERα. E2 induced a marked decrease in the invasive activity of MCF-7 and BT-474 cells but had no effect on BCM-1 cells, which had virtually no ERα. Consistent with these results, the ER-mediated Nm23-H1 promoter activity was inhibited 3-fold by the E2 antagonist, ICI 182,780. Deletion analysis of the promoter region of the Nm23-H1 gene identified a positive estrogen-responsive element located in −108/−94. ER protein bound specifically to the −108/−79 fragment with high avidity. These results indicate that E2, acting through ERα, activated transcription of the Nm23-H1 gene via a positive estrogen-responsive element in the promoter region of the gene. These results suggest that E2 could suppress tumor metastasis by activating the expression of the Nm23-H1 gene.
Ghrelin and Leptin Pulse Discharge in Fed and Fasted Rats
Authors: Bagnasco Michela , Kalra Pushpa S. , Kalra Satya P.
Published: January 2014

Ghrelin stimulates and leptin inhibits appetite by modulating neuropeptide Y (NPY) signaling in the hypothalamus. Analysis of plasma ghrelin and leptin by sensitive radioimmunoassays showed that the two peripheral hormones are secreted in pulsatile fashion in rats consuming ad libitum rat chow. Fasting augmented all parameters of ghrelin pulsatile secretion and diminished leptin secretion by selectively attenuating the pulse amplitude; concomitantly it produced synchrony in ghrelin and leptin pulse discharge. These studies imply that a synchronous leptin restraint and ghrelin stimulus on NPYergic signaling may underlie robust appetitive drive.
Targeted Deletion of the PRL Receptor: Effects on Islet Development, Insulin Production, and Glucose Tolerance
Authors: Freemark Michael , Avril Isabelle , Fleenor Don , Driscoll Phyllis , Petro Ann , Opara Emmanuel , Kendall Will , Oden Jon , Bridges Spencer , Binart Nadine , Breant Bernadette , Kelly Paul A.
Published: January 2014

PRL and placental lactogen (PL) stimulate β-cell proliferation and insulin gene transcription in isolated islets and rat insulinoma cells, but the roles of the lactogenic hormones in islet development and insulin production in vivo remain unclear. To clarify the roles of the lactogens in pancreatic development and function, we measured islet density (number of islets/cm2) and mean islet size, β-cell mass, pancreatic insulin mRNA levels, islet insulin content, and the insulin secretory response to glucose in an experimental model of lactogen resistance: the PRL receptor (PRLR)-deficient mouse. We then measured plasma glucose concentrations after ip injections of glucose or insulin. Compared with wild-type littermates, PRLR-deficient mice had 26–42% reductions (P < 0.01) in islet density and β-cell mass. The reductions in islet density and β-cell mass were noted as early as 3 wk of age and persisted through 8 months of age and were observed in both male and female mice. Pancreatic islets of PRLR-deficient mice were smaller than those of wild-type mice at weaning but not in adulthood. Pancreatic insulin mRNA levels were 20–30% lower (P < 0.05) in adult PRLR-deficient mice than in wild-type mice, and the insulin content of isolated islets was reduced by 16–25%. The insulin secretory response to ip glucose was blunted in PRLR-deficient males in vivo (P < 0.05) and in isolated islets of PRLR-deficient females and males in vitro (P < 0.01). Fasting blood glucose concentrations in PRLR-deficient mice were normal, but glucose levels after an ip glucose load were 10–20% higher (P < 0.02) than those in wild-type mice. On the other hand, the glucose response to ip insulin was normal. Our observations establish a physiologic role for lactogens in islet development and function.
Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytosolic T3 Binding Protein as a Regulator for T3-Mediated Transactivation
Authors: Mori Jun-ichirou , Suzuki Satoru , Kobayashi Mutsuhiro , Inagaki Takeshi , Komatsu Ai , Takeda Teiji , Miyamoto Takahide , Ichikawa Kazuo , Hashizume Kiyoshi
Published: January 2014

Nicotinamide adenine dinucleotide phosphate (NADPH)- dependent cytosolic T3 binding protein (CTBP) plays a role in the regulation of nuclear transport of T3in vitro. However, it is not known whether CTBP regulates the T3 action. In this study, we examined the effects of CTBP on cellular translocation of T3 and on transcriptional activation using established CTBP-expressing CHO or GH3 cells.

The expression of CTBP increased cellular and nuclear uptake of T3 in the CTBP-expressing cells. The efflux rate was decreased by induction of CTBP. Efflux from nuclei also inhibited by induction of CTBP.

Expression of CTBP suppressed the T3-regulated luciferase activity in GH3 cells. Suppression was observed to be related to the expression level of CTBP. T3 induction of rat GH mRNA was lower in the cells expressing CTBP than that in CTBP-null cells.

These results suggest that CTBP regulates the T3-induced gene expression, with which an increase in the nuclear content of the T3 is associated. Because we observed that a part of CTBP could be transported into nuclei and that acceptor protein for CTBP is present in nuclei as previously reported, interaction of CTBP with certain proteins, including transcription factors or nuclear T3 receptor, may contribute to the regulation.
Plasma Leptin Concentration in Fetal Sheep during Late Gestation: Ontogeny and Effect of Glucocorticoids
Authors: Forhead A. J. , Thomas L. , Crabtree J. , Hoggard N. , Gardner D. S. , Giussani D. A. , Fowden A. L.
Published: January 2014

The ontogeny and developmental control of plasma leptin concentration in the fetus are poorly understood. The present study investigated plasma leptin concentration in chronically catheterized sheep fetuses near term, and in neonatal and adult sheep. The effect of glucocorticoids on plasma leptin in utero was examined by fetal adrenalectomy and exogenous cortisol or dexamethasone infusion. In intact, untreated fetuses studied between 130 and 140 d (term, 145 ± 2 d), plasma leptin concentration increased in association with the prepartum cortisol surge. Positive relationships were observed between plasma leptin in utero and both gestational age and plasma cortisol. Plasma leptin was also inversely correlated with fetal paO2. The ontogenic rise in plasma leptin was abolished by fetal adrenalectomy. In intact fetuses at 123–127 d, plasma leptin was increased by infusions of cortisol (3–5 mg kg−1d−1, +127 ± 21%) for 5 d and dexamethasone (45–60 μg kg−1d−1, +268 ± 61%) for 2 d. However, the cortisol-induced rise in plasma leptin was transient; by the fifth day of infusion, plasma leptin was restored to within the baseline range. These findings show that, in the sheep fetus, an intact adrenal gland is required for the normal ontogenic rise in plasma leptin near term. Furthermore, fetal treatment with exogenous and endogenous glucocorticoids increases circulating leptin concentration in utero.
Osteoblastic Response to the Defective Matrix in the Osteogenesis Imperfecta Murine (oim) Mouse
Authors: Kalajzic I. , Terzic J. , Rumboldt Z. , Mack K. , Naprta A. , Ledgard F. , Gronowicz G. , Clark S. H. , Rowe D. W.
Published: January 2014

This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.
Immunohistochemical Localization of Microsomal PGE Synthase-1 and Cyclooxygenases in Male Mouse Reproductive Organs
Authors: Lazarus Michael , Munday Craig J. , Eguchi Naomi , Matsumoto Shigeko , Killian Gary J. , Kubata Bruno Kilunga , Urade Yoshihiro
Published: January 2014

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in the male mouse reproductive organs. Northern blotting revealed that the mPGES-1 mRNA was expressed intensely in the epididymis and weakly in the lung, spleen, skin, kidney, colon, and brain. In the male reproductive tract, the expression of mPGES-1 increased from the testis to the cauda epididymis and was highest in the vas deferens when examined by Northern blotting, RT-PCR, and Western blotting. By immunohistochemistry, mPGES-1 was detected in Leydig cells of the testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In addition, the caput and cauda regions of the epididymis and the vas deferens in this order showed a progressive increase in the expression of COX-1 mRNA and immunoreactivity, whereas COX-2 was dominantly expressed in the vas deferens. COX-1 was localized in epithelial cells of the caput, corpus and cauda epididymis and of the vas deferens, and COX-2 was evident in epithelial cells of the distal cauda epididymis and vas deferens. These results show that mPGES-1 is expressed coordinately with COX-1 and COX-2 and is involved in PGE2 production in male genital organs.
ER Function in the Adult Male Rat: Short- and Long-Term Effects of the Antiestrogen ICI 182,780 on the Testis and Efferent Ductules, without Changes in Testosterone
Authors: Oliveira Cleida A. , Zhou Qing , Carnes Kay , Nie Rong , Kuehl David E. , Jackson Gary L. , Franca Luiz R. , Nakai Masaaki , Hess Rex A.
Published: January 2014

Male rats, 30 d old, were treated with the antiestrogen ICI 182,780 (3–150 d) to determine sequences of events leading to testicular atrophy and infertility. Plasma testosterone and LH concentrations were unchanged. ICI 182,780 induced dilation of efferent ductules as early as 3 d post treatment, and the dilation increased over time, resulting in an overall increase of 200% in tubule diameter. A gradual reduction in height of the ductule epithelium was observed; however, the microvilli height increased up to d 73 but subsequently decreased. A transient increase in lysosomes in nonciliated cells was seen from d 15 to d 100. Testicular weight increased by d 45 and seminiferous tubules were dilated by d 52. These effects on testes persisted until d 100, but on d 150 the weight decreased and severe atrophy was observed. These testicular effects were probably owing to accumulation of fluid following inhibition of reabsorption in the efferent ductules, similar to the ER-α knockout mouse. In agreement with this conclusion, there was a decrease in Na+-H+ exchanger-3 mRNA and protein, which is consistent with previous studies showing that ER is required for expression of Na+-H+ exchanger-3 and ultimately fluid reabsorption in the efferent ductules.
Complex 5′ Genomic Structure of the Human Prolactin Receptor: Multiple Alternative Exons 1 and Promoter Utilization
Authors: Hu Zhang-Zhi , Zhuang Li , Meng Jianping , Tsai-Morris Chon-Hwa , Dufau Maria L.
Published: January 2014

Transcription of the prolactin receptor (PRLR) is under the control of multiple promoters. Following the recent demonstration of the human non-coding exon 1, hE1N (hE1N1) and the generic exon 1 hE13, we have identified their promoters and characterized four other novel human exons 1 (hE1N2–5) that are alternatively spliced to a common non-coding exon 2 in human tissues and breast cancer cells. Genomic regions containing these exons, and 5′-flanking and intronic sequences, were determined and their order was established in chromosome 5p14-13. Promoters utilized in the transcription of previously characterized PRLR exons 1 species hE13 (hPII) and hE1N1 (hPN1) were found to employ distinct mechanisms for controlling hPRLR transcription. hPIII requires C/EBPβ and Sp1/Sp3 for basal transcriptional activity, while hPN1 activity is conferred by domains containing an Ets element and an NR half-site. The complex promoter control system that governs transcription of the hPRLR in multiple tissues is of relevance for studies on the regulation of PRLR expression in physiological and pathological states.
Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation
Authors: Trebble P. , Matthews L. , Blaikley J. , Wayte A. W. O. , Black G. C. M. , Wilton A. , Ray D. W.
Published: January 2014

J Clin Endocrinol Metab, 10.1210/jc.2010-0705
Adipose Tissue Has Aberrant Morphology and Function in PCOS: Enlarged Adipocytes and Low Serum Adiponectin, but not Circulating Sex Steroids, Are Strongly Associated with Insulin Resistance
Authors: Mannerås-Holm Louise , Leonhardt Henrik , Kullberg Joel , Jennische Eva , Odén Anders , Holm Göran , Hellström Mikael , Lönn Lars , Olivecrona Gunilla , Stener-Victorin Elisabet , Lönn Malin
Published: January 2014

Context: Comprehensive characterization of the adipose tissue in women with polycystic ovary syndrome (PCOS), over a wide range of body mass indices (BMIs), is lacking. Mechanisms behind insulin resistance in PCOS are unclear.

Objective: To characterize the adipose tissue of women with PCOS and controls matched pair-wise for age and BMI, and to identify factors, among adipose tissue characteristics and serum sex steroids, that are associated with insulin sensitivity in PCOS.

Design/Outcome measures: Seventy-four PCOS women and 31 controls were included. BMI was 18-47 (PCOS) and 19-41 kg/m2 (controls). Anthropometric variables, volumes of subcutaneous/visceral adipose tissue (magnetic resonance imaging; MRI), and insulin sensitivity (clamp) were investigated. Adipose tissue biopsies were obtained to determine adipocyte size, lipoprotein lipase (LPL) activity, and macrophage density. Circulating testosterone, free testosterone, free 17β-estradiol, SHBG, glycerol, adiponectin, and serum amyloid A were measured/calculated.

Results: Comparison of 31 pairs revealed lower insulin sensitivity, hyperandrogenemia, and higher free 17β-estradiol in PCOS. Adipose tissue volumes/distribution did not differ in the groups, but PCOS women had higher waist-to-hip ratio, enlarged adipocytes, reduced adiponectin, and lower LPL activity. In regression analysis, adipocyte size, adiponectin, and waist circumference were the factors most strongly associated with insulin sensitivity in PCOS (R2=0.681, P < 0.001).

Conclusions: In PCOS, adipose tissue has aberrant morphology/function. Increased waist-to-hip ratio indicates abdominal/visceral fat accumulation, but this is not supported by MRI. Enlarged adipocytes and reduced serum adiponectin, together with a large waistline, rather than androgen excess, may be central factors in the pathogenesis/maintenance of insulin resistance in PCOS.
Bone Mineral Density and Testicular Failure: Evidence for a Role of Vitamin D 25-Hydroxylase in Human Testis
Authors: Foresta Carlo , Strapazzon Giacomo , De Toni Luca , Perilli Lisa , Di Mambro Antonella , Muciaccia Barbara , Sartori Leonardo , Selice Riccardo
Published: January 2014

Working Hypothesis:

Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets.

Objective:

Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients.

Design, Patients, Setting:

Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation.

Methods:

CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis.

Results:

We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05).

Conclusions:

Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.
Urocortin Expression in Human Pituitary Gland and Pituitary Adenoma
Authors: Iino Kazumi , Sasano Hironobu , Oki Yutaka , Andoh Noriaki , Shin Ryong-Woon , Kitamoto Tetsuyuki , Totsune Kazuhito , Takahashi Kazuhiro , Suzuki Hiroyoshi , Nagura Hiroshi , Yoshimi Teruya
Published: January 2014

Urocortin is a recently identified neuropeptide of the CRF family in the mammalian brain, but its expression in human tissue has been little studied. In this study, we examined urocortin expression in human anterior pituitary gland and pituitary adenomas by RIA, high performance liquid chromatography, immunohistochemistry, messenger ribonucleic acid (mRNA) in situ hybridization, and reverse transcriptase-PCR. Immunoreactive urocortin concentrations in normal pituitary tissue extract were 103.25 ± 39.05 ng/g wet wt (mean ± sem; n = 4), and their levels were all significantly higher than those in other portions of central nervous system of the same subjects. High performance liquid chromatography analysis of human pituitary extract demonstrated a single peak corresponding to that of the expected chromatographic mobility of synthetic human urocortin-(1–40). Urocortin-immunoreactive cells were detected in the anterior pituitary gland. Neither urocortin-immunoreactive nerve fibers nor cells were detected in the posterior lobe. Immunostaining in serial mirror tissue sections revealed that 76.55 ± 3.06% of urocortin-immunoreactive cells expressed GH immunoreactivity, whereas 22.25 ± 3.02% and less than 1% of urocortin-immunoreactive cells expressed PRL and ACTH, respectively. mRNA hybridization signals of urocortin were also detected in urocortin-immunopositive pituitary cells. The reverse transcriptase-PCR analysis demonstrated a 145-bp RNA band corresponding to that of the expected length of urocortin in all cases of normal pituitary glands examined (n = 3). We also immunostained urocortin in 52 cases of human anterior pituitary adenomas, including GH-producing adenomas (n = 14), ACTH-producing adenomas (n = 13), PRL-producing adenomas (n = 11), and nonfunctioning hormonally inactive adenomas (n = 14). No urocortin immunoreactivity was detected in these adenoma cells, except for one case of GH-producing adenoma and one case of nonfunctioning adenoma. We also performed mRNA in situ hybridization in 27 adenomas. No hybridization signals were detected in these adenomas, except in two cases. The results described above indicated that urocortin is synthesized in human anterior pituitary cells and may play an important role in biological features of normal pituitary gland, possibly as an autocrine or a paracrine regulator.
Resistance of Gonadotropin Releasing Hormone Drive to Sex Steroid-Induced Suppression in Hyperandrogenic Anovulation1
Authors: Daniels Tammy L. , Berga Sarah L.
Published: January 2014

Women with hyperandrogenic anovulation (HAA) exhibit increased GnRH drive, as evidenced by a faster LH pulse frequency that slows in response to progestin-induced opioidergic tone. To determine whether increased GnRH-LH drive in HAA reflects altered sex steroid exposure caused by chronic anovulation or is an intrinsic hypothalamic attribute, we compared the pulsatile LH response to oral contraceptive (OC)-induced suppression in seven women with HAA, with that of seven eumenorrheic women (EW). LH levels were determined at 10-min intervals for 12 h after 19–21 days of OC use and 5–7 days after cessation. Testosterone, androstenedione, estradiol, FSH, and LH levels were determined at weekly intervals before, during, and after OC use.

LH pulse number/12 h was higher (P < 0.001) in HAA during and after OCs, when compared with that of EW. Mean LH was increased in HAA before, during, and after OCs. Testosterone, androstenedione, and estradiol levels were higher in HAA before OCs, but they decreased to similar levels during OC use in both groups. FSH concentrations were similar before and during OCs but rose more after cessation of OCs in EW. These findings indicate that GnRH drive in HAA is resistant to OC-induced suppression and, therefore, could be an intrinsic hypothalamic attribute.
Identification of Constitutively Activating Somatic Thyrotropin Receptor Mutations in a Subset of Toxic Multinodular Goiters
Authors: Holzapfel Hans-Peter , Führer Dagmar , Wonerow Peter , Weinland Gerhard , Scherbaum Werner A. , Paschke Ralf , Führer-Sakel Dagmar
Published: January 2014

Constitutively activating mutations in the TSH receptor (TSHR) gene and in the Gsα gene are frequent molecular causes for solitary toxic nodules of the thyroid. However, the etiology of toxic multinodular goiter is still largely unknown. Therefore, DNA from nodular and quiescent surrounding tissue of six patients with toxic multinodular goiters was screened for mutations in exons 9 and 10 of the TSHR gene and exons 7–10 of the Gsα gene by direct automated sequencing.

In one patient, two different somatic TSHR mutations were identified in two different toxic nodules (L632I and F631L). In another patient, two different toxic nodules harbored the same TSHR mutation (I630L), whereas only one TSHR mutation (F631L) was identified in one of the two toxic nodules of an additional patient. In the other three patients, no mutations could be found in exons 9 and 10 of the TSHR gene or in exons 7–10 of the Gsα gene.

Our results demonstrate that not only solitary toxic adenomas but also toxic multinodular goiters can be caused by constitutively activating mutations of the TSHR. In addition to mutations in the TSHR and possibly in Gsα, there are probably other still unknown mechanisms that cause hot nodules in toxic multinodular goiters.
Recombinant Thyroid Peroxidase-Specific Fab Converted to Immunoglobulin G (IgG) Molecules: Evidence for Thyroid Cell Damage by IgG1, but Not IgG4, Autoantibodies1
Authors: Guo Jin , Jaume Juan Carlos , Rapoport Basil , McLachlan Sandra M.
Published: January 2014

A recombinant autoantibody Fab (SP1.4) to thyroid peroxidase (TPO), cloned from intrathyroidal B cell immunoglobulin genes, interacts with an epitope on TPO recognized by all patients with autoimmune thyroid disease. To compare the biological properties of IgG1 and IgG4 TPO autoantibodies, we converted Fab SP1.4 to full-length immunoglobulins. The SP1.4 heavy and κ light chain variable region genes, spliced by overlap PCR to a mammalian signal peptide, were transferred to expression vectors for human IgG1, IgG4, and κ L chains. Plasmids containing the IgG1 (or IgG4) heavy chain and the κ L chain were cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity purified from medium using protein G. Their subclass specificities were confirmed by enzyme-linked immunosorbent assay and fluorometry after binding to Chinese hamster ovary cells expressing cell surface TPO. Further confirmation of SP1.4 Fab conversion to full-length molecules was the ability of protein A to precipitate IgG1-SP and IgG4-SP complexed to [125I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab SP1.4 had similar high affinities for TPO (Kd = ∼2× 10−10 mol/L). Complexes of [125I]TPO and IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells (PBMC), but not to a B cell line. Flow cytometry demonstrated Fc receptors FcγRI, FcγRII, and FcγRIII on PBMC, but only FcγRII on the B cell line. Together, these data indicate that IgG1-SP/TPO complexes bind to either FcγRI on monocytes or RIII on natural killer cells. In assays for antibody-dependent cytotoxicity using PBMC, 51Cr release was higher for thyroid cells preincubated with IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone (−0.7%). No specific 51Cr release was observed when either fibroblasts or Chinese hamster ovary cells expressing cell surface TPO were used as target cells.

In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.
Nonassociation of Estrogen Receptor Genotypes with Bone Mineral Density and Estrogen Responsiveness to Hormone Replacement Therapy in Korean Postmenopausal Women
Authors: Han Ki Ok , Moon In Gul , Kang Young Soon , Chung Ho Yeon , Min Hun Ki , Han In Kwon
Published: January 2014

Hormone replacement therapy (HRT) prevents bone loss in postmenopausal women, but some women are resistant to therapy. A recently reported case of severe estrogen resistance caused by a germ-line mutation at the estrogen receptor (ER) gene locus suggests the possibility that other variants of the ER gene could be responsible for resistance to HRT and could also be an answer to the heritable components of bone density. Three restriction fragment length polymorphisms (RFLPs) at the ER gene locus, represented as BstUI (or B variant), PvuII, and XbaI, and their relationship to bone mineral density (BMD) and estrogen responsiveness to HRT were examined in 248 healthy postmenopausal women, aged 41–68 yr (mean± sd, 52.0 ± 4.6 yr) in Korea. The BstUI restriction site was not found in Korean women. The distribution of the PvuII and XbaI RFLPs was as follows: PP, 35 (14.1%); Pp, 136 (54.8%); pp, 77 (31.1%); and XX, 18 (7.3%); Xx, 72 (29.0%); and xx, 158 (63.7%), respectively (capital letters signify the absence of and lower case letters signify the presence of the restriction site of each RFLP). There was no significant relation between ER genotypes and z score values of lumbar spine BMD. Also, no significant genotypic differences were found in the change in lumbar spine BMD and those in biochemical markers before and after 1 yr of HRT. These data indicate no significant effects of ER genotypes on BMD and estrogen responsiveness after HRT.
Serum Leptin Concentrations in Women with Polycystic Ovary Syndrome
Authors: Rouru Juha , Anttila Leena , Koskinen Pertti , Penttilä Tuula-Anneli , Irjala Kerttu , Huupponen Risto , Koulu Markku
Published: January 2014

The role of gonadotropins, androgens, and insulin in the regulation of circulating leptin levels is obscure. In order to clarify the relationships of these parameters we studied serum leptin levels in 19 healthy control subjects and in 35 hyperandrogenic and hyperinsulinemic patients with polycystic ovary syndrome (PCOS).

Serum leptin concentrations did not differ significantly between PCOS patients and control subjects. When PCOS and control groups were analyzed together by univariate analysis, serum leptin was positively correlated with body mass index (BMI), body weight, serum insulin, serum triglyceride, and serum free testosterone concentrations. Serum leptin was inversely correlated with serum sex hormone binding globulin (SHBG) concentrations. There were no significant correlations between serum leptin and testosterone, androstenedione, or gonadotropin concentrations. Serum insulin, triglyceride, and free testosterone concentrations were positively correlated, and serum SHBG was negatively correlated with BMI. However, when BMI on one hand and serum insulin, triglyceride, free testosterone, or SHBG on other hand were used as independent variables in the partial correlation analysis with leptin, BMI turned out to be the variable primarily responsible for all of the correlations with leptin.

In conclusion, the concept that circulating leptin levels would be different in PCOS patients than in regularly menstruating control subjects is not supported by our data.
Fifty Years of Experience with Propylthiouracil-Associated Hepatotoxicity: What Have We Learned?1
Authors: Williams Katherine V. , Nayak Sunil , Becker Dorothy , Reyes Jorge , Burmeister Lynn A.
Published: January 2014

The aim of this study was to determine the optimal management of patients with propylthiouracil (PTU) hepatotoxicity.

A MEDLINE search for English language cases of PTU hepatotoxicity between 1966 and April 1996 was performed, and additional cases were cross-referenced. Twenty-seven cases were selected based on the availability of information on patient management after the onset of hepatotoxicity. Eighty-five percent of the selected cases met this criterion. A detailed summary of the management of two cases of PTU hepatotoxicity at our institutions is also provided.

Although most patients recovered once PTU was stopped, seven patients died. Patients with PTU hepatotoxicity who survived were more likely to have received 131I during the course of their illness than those who died (P < 0.03, by Fisher’s exact test). In our two patients, hyperbilirubinemia was linearly associated with progressively decreasing T4 levels (r = 0.91; P < 0.001) despite the presence of clinical thyrotoxicosis in one of the patients. These findings demonstrate the need for appropriate clinical evaluation and treatment of thyroid disease during the course of hepatotoxicity. Additionally, we report the first pediatric patient with PTU hepatotoxicity to undergo liver transplantation. The emerging role of liver transplantation in these patients is discussed.
Leptin Concentrations in the Polycystic Ovary Syndrome1
Authors: Mantzoros Christos S. , Dunaif Andrea , Flier Jeffrey S.
Published: January 2014

The polycystic ovary syndrome (PCOS) is characterized by menstrual disturbances, chronic anovulation and hyperandrogenism and is associated with insulin resistance and hyperinsulinemia. Leptin, the product of the ob gene, is an adipocyte-secreted molecule that signals the magnitude of energy stores to the brain and has been recently shown to have important effects on the reproductive axis of rodents. To assess the potential contribution of leptin to the pathogenesis of PCOS, we measured leptin levels in 24 obese women with PCOS and 12 weight- and age-matched controls and determined whether alterations in hyperinsulinemia produced by administration of the insulin-sensitizing agent troglitazone had an effect on serum leptin levels. Leptin concentrations at baseline were not different in women with PCOS (38.1 ± 2.15 ng/mL) and controls (33.12 ± 2.39 ng/mL). Moreover, leptin concentrations remained unchanged after treatment with troglitazone (38.1 ± 2.15 vs. 39.21 ± 2.65 ng/mL). Baseline leptin correlated strongly with body mass index in both controls (r = 0.59; P< 0.05) and women with PCOS (r = 0.70; P = 0.0004). Leptin levels were not associated with baseline insulin, testosterone, non-sex hormone-binding globulin (SHBG)-bound testosterone, dehydroepiandrosterone sulfate, estradiol, or SHBG. Finally, despite significantly reduced insulin, non-SHBG-bound testosterone, and estradiol levels after troglitazone treatment of women with PCOS, their leptin levels remained unchanged. We conclude that circulating leptin levels in patients with PCOS do not differ from those in age- and weight-matched controls. Furthermore, increased circulating insulin due to insulin resistance does not appear to alter circulating leptin levels in women with PCOS.
Peak and Trough Growth Hormone Concentrations Have Different Associations with the Insulin-Like Growth Factor Axis, Body Composition, and Metabolic Parameters1
Authors: Hindmarsh P. C. , Fall C. H. D. , Pringle P. J. , Osmond C. , Brook C. G. D.
Published: January 2014

GH is secreted in a pulsatile fashion, promoting growth and anabolism. The components of the pulsatile signal involved in these diverse effects are unclear. We constructed (20-min sampling interval) and analyzed 24-h serum GH profiles in 45 adult male volunteers, 59.4–69.9 yr old, body mass index (BMI) 21.9–36.5 Kg/m2, using Fourier transformation and a concentration distribution analysis that determines the concentration at or below which the serum GH concentrations in the 24-h profile spend a percentage of the total time. The observed concentrations (OC) below which 95% and 5% of the values in the time series lie [lsb]OC95 (peaks) and OC5 (troughs)] and mean 24-h serum GH concentrations were related to measures of the insulin-like growth factor (IGF) family, parameters of body composition, fasting insulin and cholesterol measures, and GH-binding protein concentrations.

Mean 24-h serum GH concentrations ranged between 0.19 and 2.15 mU/L (1μ g/L = 2.6 mU/L). Pulse periodicity was between 180 and 200 min. There was a positive relationship between peak GH levels and serum IGF-1 and IGFBP-3 levels (r = 0.39; P = 0.009 and r = 0.32; P = 0.03, respectively). GH trough levels were unrelated to these measures of the IGF family. In contrast, GH troughs were related inversely to BMI (r = −0.31; P = 0.04) and waist-hip ratio (r = −0.4; P = 0.006). Peak GH levels were not related to these measures. Factors known to influence these measures, fasting insulin concentration, or cortisol secretion did not alter the trough GH relationship in multiple regression analysis. All GH parameters were related inversely to fasting insulin concentration. Although GH parameters were related inversely to cholesterol and low-density lipoprotein-cholesterol, this effect disappeared when age and fasting insulin levels were introduced into the regression. GH-binding protein levels related most strongly to BMI (r = 0.60; P < 0.001), with no effect of any GH parameter observed in multiple regression analysis. These results suggest that the peak values of a GH concentration profile may influence the IGF axis, whereas trough values may influence body composition and metabolic parameters of GH action.
Expression of Estrogen Receptor-β in Human Breast Tumors
Authors: Dotzlaw Helmut , Leygue Etienne , Watson Peter H. , Murphy Leigh C.
Published: January 2014

The expression of a recently described novel estrogen receptor, ER-β, was detected in several human breast tumor biopsy samples and several human breast epithelial cell lines using reverse transcription and polymerase chain reaction (RT-PCR) analysis. Cloning and sequencing of the PCR product from a breast tumor confirmed the identity of the sequence with that of the ER-β mRNA previously reported in human testis. The expression of ER-β was not correlated with that of ER-α, and both ER-α positive and ER-α negative cell lines expressed ER-β mRNA. However, some breast tumors and some cell lines coexpress ER-β and ER-α mRNA. Our data support a possible role for ER-β in human breast cancer.