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Sample records for expressing human fsh

  1. FSH-Receptor Isoforms and FSH-dependent Gene Transcription in Human Monocytes and Osteoclasts

    PubMed Central

    Robinson, Lisa J; Tourkova, Irina; Wang, Yujuan; Sharrow, Allison C; Landau, Michael S; Yaroslavskiy, Beatrice B; Li, Sun; Zaidi, Mone; Blair, Harry C

    2010-01-01

    Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8–10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes or osteoclasts transcribed TNFα in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2 hours in 25 ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFα and TSG-6, increased 2–3 fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms. PMID:20171950

  2. Modulation of gene expression in small follicle porcine granulosa cells by human follicle stimulating hormone (hFSH)

    SciTech Connect

    Calvo, F.O.; Ryan, R.J.; Woloschak, G.E.

    1986-03-01

    Small follicle (1-3 mm) porcine granulosa cells (SFPGF) were isolated by puncture, aspiration and cultured under standard conditions in DMEM, HEPES, BSA, MIX. At the start of culture, cells were stimulated with 100ng hFSH/ml. At various times afterwards total cellular RNA was prepared using guanidine-hydrochloride solubilization, phenol extraction and precipitation from 3M NaOAc, pH 6.0. RNA was 5'-end labelled with /sup 32/P in a kinase reaction and hybridized to an excess of clone-specific DNA immobilized on nitrocellulose filters using stringent hybridization and wash conditions. After autoradiography the RNA hybridized to the DNA blot filter were quantitated by microdensitometry. Hybridization to parent plasmid was negative. RNA derived from control cultures showed patterns of hybridization similar to those obtained from freshly obtained cells. Results of these experiments demonstrate hFSh induction of RNA specific for transferrin receptor, ..cap alpha..-interferon, H-ras, and K-ras. Increased RNA levels were apparent within 10 min of treatment and had declined by 180 min. Expression of actin, p53 and for RNAs declined by 10 min of hFSH addition but was enhanced by 160 min. Levels of ..beta..-interferon, myc, mos, abl and yb RNAs were not detectable under these conditions. These results demonstrate specific gene modulation in SFPGC cultured with hFSH.

  3. Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

    PubMed

    Wang, Hong; Chen, Xi; Zhang, Xiaoxi; Zhang, Wei; Li, Yan; Yin, Hongrui; Shao, Hong; Chen, Gang

    2016-03-04

    Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

  4. FSH stimulates lipid biosynthesis in chicken adipose tissue by upregulating the expression of its receptor FSHR.

    PubMed

    Cui, Huanxian; Zhao, Guiping; Liu, Ranran; Zheng, Maiqing; Chen, Jilan; Wen, Jie

    2012-05-01

    Transcripts and protein for follicle-stimulating hormone receptor (FSHR) were demonstrated in abdominal adipose tissue of female chickens. There was no expression of the Fsh gene, but FSH and FSHR colocalized, suggesting that FSH was receptor bound. Partial correlations indicted that changes in abdominal fat (AF) content were most directly correlated with Fshr mRNA expression, and the latter was directly correlated with tissue FSH content. These relationships were consistent with FSH inducing Fshr mRNA expression and with the finding that FSH influenced the accumulation of AF in chickens, a novel role for the hormone. Chicken preadipocytes responded linearly to doubling concentrations of FSH in Fshr mRNA expression and quantities of FSHR and lipid, without discernable effect on proliferation. Cells exposed to FSH more rapidly acquired adipocyte morphology. Treatment of young chickens with chicken FSH (4 mIU/day, subcutaneous, days 7-13) did not significantly decrease live weight but increased AF weight by 54.61%, AF as a percentage of live weight by 55.45%, and FSHR transcripts in AF by 222.15% (2 h after injection). In cells stimulated by FSH, genes related to lipid metabolism, including Rdh10, Dci, RarB, Lpl, Acsl3, and Dgat2, were expressed differentially, compared with no FSH. Several pathways of retinal and fatty acid metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling changed. In conclusion, FSH stimulates lipid biosynthesis by upregulating Fshr mRNA expression in abdominal adipose tissue of chickens. Several genes involved in fatty acid and retinal metabolism and the PPAR signaling pathway mediate this novel function of FSH.

  5. Exogenous administration of recombinant human FSH does not improve germ cell survival in human prepubertal xenografts.

    PubMed

    Van Saen, Dorien; Goossens, Ellen; Haentjens, Patrick; Baert, Yoni; Tournaye, Herman

    2013-03-01

    In a previous study, meiotic activity was observed in human intratesticular xenografts from peripubertal patients. However, full spermatogenesis could not be established. The present study aimed to evaluate whether the administration of recombinant human FSH could improve the spermatogonial survival and the establishment of full spermatogenesis in intratesticular human xenografts. Human testicular tissue was obtained from six boys (aged 2.5-12.5years). The testicular biopsy was fragmented and one fragment of 1.5-3.0mm(3) was transplanted to the testis of immunodeficient nude mice. Transplanted mice were assigned to different experimental groups to enable evaluation of the effects of FSH administration and freezing. The structural integrity of the seminiferous tubules, the spermatogonial survival and the presence of differentiated cells were evaluated by histology and immunohistochemistry. Freezing or administration of FSH did not influence tubule integrity and germ cell survival in human xenografts. Meiotic germ cells were observed in the xenografts. More tubules containing only Sertoli cells were observed in frozen-thawed grafts, and more tubules with meiotic cells were present in fresh grafts. There was no clear influence of FSH treatment on meiotic differentiation. Administration of FSH did not improve the establishment of full spermatogenesis after intratesticular tissue grafting. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  6. FSH and eCG impact follicles development and expression of ovarian FSHR and caspase-9 in mice.

    PubMed

    Wei, S; Gong, Z; Guo, H; Zhang, T; Ma, Z

    2017-01-01

    The study aimed to investigate the effects of FSH and eCG on the ovarian and follicular development, expression levels of FSHR and caspase-9 of ovaries in vivo. One hundred and five prepuberty mice were allocated into FSH-1, FSH-2, FSH-3, eCG-1, eCG-2, eCG-3 groups and control group (CG). Mice in FSH-1, FSH-2 and FSH-3 were intramuscularly injected with 5, 10 and 20 IU FSH twice (on day 0 and 4), respectively. Mice in eCG-1, eCG-2 and eCG-3 were intraperitoneally injected with 10, 20 and 40 IU eCG on day 0 and 4. Mice in the CG were injected with 0.5 ml normal saline on day 0 and 4. Left and right ovaries of each mouse were dissected aseptically on days 7, 14 and 21, respectively. The results showed that on days 14 and 21 the ovarian sizes and follicle numbers of FSH-3 and eCG-3 groups were greater than CG (P<0.05). FSHR mRNA of FSH-2 and eCG-1 were higher than CG on days 14 and 21 (P<0.05). FSHR proteins of FSH-3 were higher than CG on days 14 and 21 (P<0.05). Caspase-9 mRNA in FSH and eCG groups was less than CG. There were positive correlations between follicle numbers and FSH and eCG doses. FSHR protein expressions had positive correlations between ovarian weights and sizes of ovary and follicle numbers (r=0.971, P<0.05) in FSH-treated mice. Serum FSH concentrations of FSH-2, FSH-3, eCG-2 and eCG-3 groups were greater than that of CG. In conclusion, eCG and FSH promoted the ovarian development, follicle genesis, FSH secretion, FSHR mRNA and protein expressions in ovaries of mice. FSH and eCG inhibited the expression of ovarian caspase-9 mRNA.

  7. Expression profiles of Fsh-regulated ovarian genes during oogenesis in coho salmon.

    PubMed

    Guzmán, José M; Luckenbach, J Adam; Yamamoto, Yoji; Swanson, Penny

    2014-01-01

    The function of follicle-stimulating hormone (Fsh) during oogenesis in fishes is poorly understood. Using coho salmon as a fish model, we recently identified a suite of genes regulated by Fsh in vitro and involved in ovarian processes mostly unexplored in fishes, like cell proliferation, differentiation, survival or extracellular matrix (ECM) remodeling. To better understand the role of these Fsh-regulated genes during oocyte growth in fishes, we characterized their mRNA levels at discrete stages of the ovarian development in coho salmon. While most of the transcripts were expressed at low levels during primary growth (perinucleolus stage), high expression of genes associated with cell proliferation (pim1, pcna, and mcm4) and survival (ddit4l) was found in follicles at this stage. The transition to secondary oocyte growth (cortical alveolus and lipid droplet stage ovarian follicles) was characterized by a marked increase in the expression of genes related to cell survival (clu1, clu2 and ivns1abpa). Expression of genes associated with cell differentiation and growth (wt2l and adh8l), growth factor signaling (inha), steroidogenesis (cyp19a1a) and the ECM (col1a1, col1a2 and dcn) peaked in vitellogenic follicles, showing a strong and positive correlation with transcripts for fshr. Other genes regulated by Fsh and associated with ECM function (ctgf, wapl and fn1) and growth factor signaling (bmp16 and smad5l) peaked in maturing follicles, along with increases in steroidogenesis-related gene transcripts. In conclusion, ovarian genes regulated by Fsh showed marked differences in their expression patterns during oogenesis in coho salmon. Our results suggest that Fsh regulates different ovarian processes at specific stages of development, likely through interaction with other intra- or extra-ovarian factors.

  8. Clinical efficacy and cost-effectiveness of HP-human FSH (Fostimon®) versus rFSH (Gonal-F®) in IVF-ICSI cycles: a meta-analysis.

    PubMed

    Gerli, Sandro; Bini, Vittorio; Favilli, Alessandro; Di Renzo, Gian Carlo

    2013-06-01

    Clinical efficacy of human-derived follicle-stimulating hormone (FSH) versus recombinant FSH (rFSH) in IVF-ICSI cycles has long been compared, but no clear evidence of the superiority of a preparation over the other has been found. Human gonadotropins have been often grouped together, but a different glycosylation may be present in each preparation, therefore influencing the specific bioactivity. To exclude confounding factors, a meta-analysis and a cost-effectiveness analysis were designed to compare effectiveness and cost-effectiveness of a specific highly purified human FSH (HP-hFSH) (Fostimon®) versus rFSH (Gonal-F®) in IVF/ICSI cycles. Research methodology filters were applied in MEDLINE, Current Contents and Web of Science from 1980 to February 2012. Eight randomized trials met selection criteria. The meta-analysis showed no significant differences between rFSH and HP-hFSH treatment in live-birth rate (odds ratio [OR] 0.84, 95% confidence interval [CI] 0.63-1.11), clinical pregnancy rate (OR 0.85, 95% CI 0.68-1.07), number of oocytes retrieved, number of mature oocytes and days of stimulation. The cost-effectiveness ratio was € 7174 in the rFSH group and € 2056 in the HP-hFSH group. HP-hFSH is as effective as rFSH in ovarian stimulation for IVF-ICSI cycles, but the human preparation is more cost-effective.

  9. Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland.

    PubMed

    Miyano, Yuki; Tahara, Shigeyuki; Sakata, Ichiro; Sakai, Takafumi; Abe, Hiroyuki; Kimura, Shioko; Kurotani, Reiko

    2014-04-01

    Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPβ, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

  10. Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells

    SciTech Connect

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Glisic, Branka; Kaisarevic, Sonja; Kovacevic, Radmila; Andric, Nebojsa

    2013-07-01

    Worldwide used herbicide atrazine is linked to reproductive dysfunction in females. In this study, we investigated the effects and the mechanism of atrazine action in the ovary using a primary culture of immature granulosa cells. In granulosa cells, follicle-stimulating hormone (FSH) activates both cyclic adenosine monophosphate (cAMP) and extracellular-regulated kinase 1/2 (ERK1/2) cascades, with cAMP pathway being more important for luteinizing hormone receptor (LHR) and aromatase (CYP19A1) mRNA expression. We report that 48 h after atrazine exposure the FSH-stimulated LHR and CYP19A1 mRNA expression and estradiol synthesis were decreased, with LHR mRNA being more sensitive to atrazine than CYP19A1 mRNA. Inadequate acquisition of LHR in the FSH-stimulated and atrazine-exposed granulosa cells renders human chorionic gonadotropin (hCG) ineffective to stimulate amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr) mRNA expression, suggesting anti-ovulatory effect of atrazine. To dissect the signaling cascade involved in atrazine action in granulosa cells, we used U0126, a pharmacological inhibitor of ERK1/2. U0126 prevents atrazine-induced decrease in LHR and CYP19A1 mRNA levels and estradiol production in the FSH-stimulated granulosa cells. ERK1/2 inactivation restores the ability of hCG to induce expression of the ovulatory genes in atrazine-exposed granulosa cells. Cell-based ELISA assay revealed that atrazine does not change the FSH-stimulated ERK1/2 phosphorylation in granulosa cells. The results from this study reveal that atrazine does not affect but requires ERK1/2 phosphorylation to cause decrease in the FSH-induced LHR and CYP19A1 mRNA levels and estradiol production in immature granulosa cells, thus compromising ovulation and female fertility. - Highlights: • Atrazine inhibits estradiol production in FSH-stimulated granulosa cells. • Atrazine inhibits LHR and Cyp19a1 mRNA expression in FSH-stimulated granulosa cells. • Atrazine

  11. Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

    SciTech Connect

    Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. )

    1991-06-01

    We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

  12. Cloning and gene expression of a cDNA for the chicken follicle-stimulating hormone (FSH)-beta-subunit.

    PubMed

    Shen, San-Tai; Yu, John Yuh-Lin

    2002-02-15

    Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein hormones that are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSH-beta in avian species. For better understanding of the phylogenic diversity and evolution of FSH molecule, we have isolated and sequenced the complete complementary DNA (cDNA) encoding chicken FSH-beta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned chicken FSH-beta cDNA consists of 2457-bp nucleotides, including 44-bp nucleotides of the 5'-untranslated region (UTR), 396 bp of the open reading frame, and an extraordinarily long 3'-UTR of 2001-bp nucleotides followed by a poly(A)((16)) tail. It encodes a 131-amino-acid precursor molecule of FSH-beta-subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the chicken FSH-beta-subunit. Four proline residues, presumably responsible for changing the backbone direction of protein structure, are conserved in chicken FSH-beta-subunit as well. The nucleotide sequence of chicken FSH-beta cDNA shows high homology with quail FSH-beta cDNA, 97% homology in the open reading frame, and 85% homology in the 3'-UTR. The deduced amino acid sequence of chicken FSH-beta-subunit shows a remarkable similarity to other avian FSH-beta-subunits, 98% homology with quail, and 93% homology with ostrich, whereas a lower similarity (66 to 70%) is noted when compared with mammalian FSH-beta-subunits. By contrast, when comparing with the beta-subunits of chicken luteinizing hormone and thyroid-stimulating hormone, the homologies are as low as 37 and 40%, respectively. FSH-beta mRNA was only expressed in pituitary gland out of various

  13. FSHbeta gene mutation in a female with delayed puberty and hypogonadism: response to recombinant human FSH.

    PubMed

    Kottler, M L; Richard, N; Chabre, O; Alain, S; Young, J

    2009-01-01

    We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary FSH that does not respond to GnRH administration. Serum inhibin B was undetectable and antimullerian hormone (AMH) was within the normal range. Ultra sound examination revealed a small uterus and small ovaries with few small follicles. We identified an homozygous 1-bp (G) deletion at codon 79 in FSHbeta gene suggesting a complete loss of function. The patient underwent studies of ovarian responsiveness to recombinant human FSH according to the following protocol: 150UI/d for five days following by 75 UI/d for 10 days. Estradiol plasma level started to increase from day 5 associated to a sharp increase of inhibine B and a decrease of LH. During the same time, we observed an excessive development of multiple follicles resulting in an arrest of the treatment to avoid hyperstimulation. The present study confirm that follicles up to 5 mm in diameter had developed in the absence of FSH and that FSH is required for the growth of follicles beyond the two-layer granulose stage.

  14. Hypo-glycosylated Human Follicle-Stimulating Hormone (hFSH21/18) is much more active in vitro than Fully-glycosylated hFSH (hFSH24)

    PubMed Central

    Bousfield, George R.; Butnev, Vladimir Y.; Butnev, Viktor Y.; Hiromasa, Yasuaki; Harvey, David J.; May, Jeffrey V.

    2014-01-01

    Hypo-glycosylated hFSH21/18 (possesses FSHβ21 and FSH18 bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH24 was prepared by combining the fully-glycosylated FSHβ24 variant with hCGα and isolating the heterodimer. The hFSH21/18 glycoform preparation was significantly smaller than the hFSH24 preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH21/18 was 9- to 26-fold more active than fully-glycosylated hFSH24 in FSH radioligand assays. Significantly greater binding of 125I-hFSH21/18 tracer than hFSH24 tracer was observed in all competitive binding assays. In addition, higher binding of hFSH21/18 was noted in association and saturation binding assays, in which twice as much hFSH21/18 was bound as hFSH24. This suggests that more ligand binding sites are available to hFSH21/18 in FSHR than to hFSH24. Hypo-glycosylated hFSH21/18 also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH24 specific binding proceeded very slowly for almost the first hour of incubation. PMID:24291635

  15. The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

    PubMed

    Chaves, Roberta N; Duarte, Ana Beatriz G; Rodrigues, Giovanna Q; Celestino, Juliana J H; Silva, Gerlane M; Lopes, Claudio Afonso P; Almeida, Anderson P; Donato, Mariana A M; Peixoto, C A; Moura, Arlindo A A; Lobo, Carlos H; Locatelli, Yann; Mermillod, Pascalle; Campello, Claudio C; Figueiredo, Jose Ricardo

    2012-09-01

    The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

  16. Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

    PubMed Central

    Dell'Aquila, Maria Elena; Caillaud, Maud; Maritato, Filippo; Martoriati, Alain; Gérard, Nadine; Aiudi, Giulio; Minoia, Paolo; Goudet, Ghylène

    2004-01-01

    The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms. PMID:15212696

  17. Characterization of the chicken follicle-stimulating hormone receptor (cFSH-R) complementary deoxyribonucleic acid, and expression of cFSH-R messenger ribonucleic acid in the ovary.

    PubMed

    You, S; Bridgham, J T; Foster, D N; Johnson, A L

    1996-11-01

    Studies were conducted to characterize the chicken (c) FSH receptor (R) cDNA, and to evaluate expression of cFSH-R mRNA in the hen ovary at known stages during follicle development. A total of 2.5 kb of nucleic acid sequence including the complete cFSH-R coding region was isolated by a combination of the reverse-transcription polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends techniques. Overall, the nucleic acid sequence homology of the cFSH-R cDNA coding region is 71.8% and 72.2% compared to the rat and bovine FSH-R, respectively, while the deduced amino acid sequence identity for the receptor protein (693 amino acids) is 71.9% and 72.4%, respectively. By comparison, the cFSH-R nucleic acid and amino acid sequences are 60.1% and 49.4% identical to the respective cLH-R sequences. Northern blot analysis detected a single 4.3-kb cFSH-R mRNA transcript, which was selectively expressed in ovarian (granulosa, theca, and stromal) tissues, but not the oviduct, adrenal, liver, muscle, or brain. As the follicle developed from the prehierarchical (6- to 8-mm diameter) to the largest preovulatory (F1 follicle) stage, cFSH-R mRNA levels progressively declined within both the granulosa and theca layers (p < 0.05). Moreover, cFSH-R mRNA levels were lower in whole atretic than in morphologically normal 3- to 5-mm follicles (p = 0.0015). The pattern of cFSH-R mRNA expression within the granulosa layer during follicle development was notably different from that of the recently reported cLH-R, in that cLH-R mRNA levels increase to become readily detectable coincident with dramatically increased steroidogenic capacity during the last few days before ovulation of the follicle. On the other hand, highest levels of cFSH-R mRNA in 6- to 8-mm (prehierarchical) follicles were consistent with a role for the cFSH-R in maintaining the viability of prehierarchical follicles and in initiating granulosa cell differentiation at the time when follicles are selected into the

  18. Fsh controls gene expression in fish both independently of and through steroid mediation.

    PubMed

    Sambroni, Elisabeth; Lareyre, Jean-Jacques; Le Gac, Florence

    2013-01-01

    The mechanisms and the mediators relaying Fsh action on testicular functions are poorly understood. Unlike in mammals, in fish both gonadotropins (Fsh and Lh) are able to efficiently stimulate steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, it is crucial to understand if Fsh effects are mediated through the production of steroids. To address this issue we performed transcriptome studies after in vitro incubations of rainbow trout testis explants in the presence of Fsh alone or in combination with trilostane, an inhibitor of Δ4- steroidogenesis. Trilostane significantly reduced or suppressed the response of many genes to Fsh (like wisp1, testis gapdhs, cldn11, inha, vt1 or dmrt1) showing that, in fish, important aspects of Fsh action follow indirect pathways and require the production of Δ4-steroids. What is more, most of the genes regulated by Fsh through steroid mediation were similarly regulated by Lh (and/or androgens). In contrast, the response to Fsh of other genes was not suppressed in the presence of trilostane. These latter included genes encoding for anti-mullerian hormone, midkine a (pleiotrophin related), angiopoietine-related protein, cyclins E1 and G1, hepatocyte growth factor activator, insulin-like growth factor 1b/3. A majority of those genes were preferentially regulated by Fsh, when compared to Lh, suggesting that specific regulatory effects of Fsh did not depend on steroid production. Finally, antagonistic effects between Fsh and steroids were found, in particular for genes encoding key factors of steroidogenesis (star, hsd3b1, cyp11b2-2) or for genes of the Igf system (igf1b/3). Our study provides the first clear evidence that, in fish, Fsh exerts Δ4-steroid-independent regulatory functions on many genes which are highly relevant for the onset of spermatogenesis.

  19. Comparison of marmoset and human FSH using synthetic peptides of the β-subunit L2 loop region and anti-peptide antibodies.

    PubMed

    Kutteyil, Susha S; Kulkarni, Bhalchandra J; Mojidra, Rahul; Joseph, Shaini; Pathak, Bhakti R; Mahale, Smita D

    2016-06-01

    Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH-FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti-peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β-subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32-50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone-receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross-species peptide and FSH in ELISA. The anti-peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH-FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  20. The effects of levonorgestrel on FSH-stimulated primary rat granulosa cell cultures through gene expression profiling are associated to hormone and folliculogenesis processes.

    PubMed

    Lira-Albarrán, Saúl; Larrea-Schiavon, Marco F; González, Leticia; Durand, Marta; Rangel, Claudia; Larrea, Fernando

    2017-01-05

    Levonorgestrel (LNG), a synthetic progestin, is used in emergency contraception (EC). The mechanism is preventing or delaying ovulation at the level of the hypothalamic pituitary unit; however, little knowledge exists on LNG effects at the ovary. The aim of this study was to identify the effects of LNG on FSH-induced 17β-estradiol (E2) production, including LNG-mediated changes on global gene expression in rat granulosa cells (GC). Isolated GC from female Wistar rats were incubated in vitro in the presence or absence of human FSH and progestins. At the end of incubations, culture media and cells were collected for E2 and mRNA quantitation. The results showed the ability of LNG to inhibit both hFSH-induced E2 production and aromatase gene expression. Microarray analysis revealed that LNG treatment affects GC functionality particularly that related to folliculogenesis and steroid metabolism. These results may offer additional evidence for the mechanisms of action of LNG as EC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Recombinant human follicle-stimulating hormone (r-hFSH) plus recombinant luteinizing hormone versus r-hFSH alone for ovarian stimulation during assisted reproductive technology: systematic review and meta-analysis

    PubMed Central

    2014-01-01

    Background The potential benefit of adding recombinant human luteinizing hormone (r-hLH) to recombinant human follicle-stimulating hormone (r-hFSH) during ovarian stimulation is a subject of debate, although there is evidence that it may benefit certain subpopulations, e.g. poor responders. Methods A systematic review and a meta-analysis were performed. Three databases (MEDLINE, Embase and CENTRAL) were searched (from 1990 to 2011). Prospective, parallel-, comparative-group randomized controlled trials (RCTs) in women aged 18–45 years undergoing in vitro fertilization, intracytoplasmic sperm injection or both, treated with gonadotrophin-releasing hormone analogues and r-hFSH plus r-hLH or r-hFSH alone were included. The co-primary endpoints were number of oocytes retrieved and clinical pregnancy rate. Analyses were conducted for the overall population and for prospectively identified patient subgroups, including patients with poor ovarian response (POR). Results In total, 40 RCTs (6443 patients) were included in the analysis. Data on the number of oocytes retrieved were reported in 41 studies and imputed in two studies. Therefore, data were available from 43 studies (r-hFSH plus r-hLH, n = 3113; r-hFSH, n = 3228) in the intention-to-treat (ITT) population (all randomly allocated patients, including imputed data). Overall, no significant difference in the number of oocytes retrieved was found between the r-hFSH plus r-hLH and r-hFSH groups (weighted mean difference −0.03; 95% confidence interval [CI] −0.41 to 0.34). However, in poor responders, significantly more oocytes were retrieved with r-hFSH plus r-hLH versus r-hFSH alone (n = 1077; weighted mean difference +0.75 oocytes; 95% CI 0.14–1.36). Significantly higher clinical pregnancy rates were observed with r-hFSH plus r-hLH versus r-hFSH alone in the overall population analysed in this review (risk ratio [RR] 1.09; 95% CI 1.01–1.18) and in poor responders (n = 1179; RR 1.30; 95% CI 1

  2. Occurrence of FSH, inhibin and other hypothalamic-pituitary-intestinal hormones in normal fertility, subfertility, and tumors of human testes.

    PubMed

    Mehta, M K; Garde, S V; Sheth, A R

    1995-01-01

    To compare the distribution of peptide hormones in presumably normal human testicular tissues and specimens exhibiting any of five pathologies. Biopsies from patients having testicular malfunctions were prepared as sections and specifically immunohistochemically stained for inhibin, FSH, serotonin, AUP, and oxytocin. Immunocytochemical studies revealed the presence of various hypophysial-pituitary-intestinal hormones, viz., FSH, inhibin, arginine vasopressin (AVP), calcitonin, serotonin, oxytocin, adrenocorticotropin (ACTH), gastrin, secretin, and somatostatin in human testicular biopsies exhibiting normal spermatogenesis, Sertoli-cell-only syndrome, spermatogenic arrest, Leydig cell hyperplasia, Leydig cell tumor, and seminoma. Intensity of immunostaining for all peptides except FSH was stronger in cases of subfertile as compared to normal testis. Intensity of immunostaining with inhibin was maximum in Leydig cell tumor. These regulatory peptides may be involved in the pathophysiology of the testes.

  3. Effect of FSH and LH hormones on oocyte maturation of buffalo and gene expression analysis of their receptors and Cx43 in maturing oocytes.

    PubMed

    Pandey, Alok; Gupta, S C; Gupta, Neelam

    2010-08-01

    Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus-oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0-6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation.

  4. Immunoradiometric assay (IRMA) for human follicle stimulating hormone (FSH) and luteinizing hormone (LH) using common avidin solid phase.

    PubMed

    Vrinda, C; Paradkar, S N; Jyotsna, N; Sivaprasad, N

    2003-01-01

    This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with 125I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0-200 mIU/mL) and analytical recovery (87-110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).

  5. Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.

    PubMed

    Dierich, A; Sairam, M R; Monaco, L; Fimia, G M; Gansmuller, A; LeMeur, M; Sassone-Corsi, P

    1998-11-10

    Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R -/- mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R -/- mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

  6. Mixed protocols: Multiple ratios of FSH and LH bioactivity using highly purified, human-derived FSH (BRAVELLE) and highly purified hMG (MENOPUR) are unaltered by mixing together in the same syringe

    PubMed Central

    Scobey, M Joseph; Raike, Elizabeth; Marshall, Dennis C

    2005-01-01

    Background The use of mixed or blended protocols, that utilize both FSH and hMG, for controlled ovarian hyperstimulation is increasing in use. To reduce the number of injections a patient must administer, many physicians instruct their patients to mix their FSH and hMG together to be given as a single injection. Therefore, the goal of this study was to definitively determine if the FSH and LH bioactivities of highly purified, human-derived FSH (Bravelle(R)) and highly purified hMG (Menopur(R)) were altered by reconstituting in 0.9% saline and mixing in the same syringe. Methods Bravelle(R) and Menopur(R) were reconstituted in 0.9% saline and mixed in a Becton Dickinson plastic syringe. The FSH and LH bioactivities of the products were determined after injecting female and male rats, respectively, with Bravelle(R), Menopur(R), or a mixture of Bravelle(R) and Menopur(R). Ratios of FSH:LH activity tested were 150:75 IU (1 vial Bravelle(R): 1 vial Menopur(R)), 300:75 IU (3 vials Bravelle(R): 1 vial Menopur(R)) or 300:225 IU (1 vial Bravelle(R): 3 vials of Menopur(R)). Results There were no statistically significant changes in either FSH or LH bioactivity that occurred after mixing Bravelle(R) with Menopur(R) in the same syringe. The theoretical vs. actual FSH bioactivity for Bravelle(R) and Menopur(R) were 75 vs. 76.58 IU/mL and 75 vs. 76.0 IU/mL, respectively. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R): 1 vial Menopur(R)), 300:75 IU (3 vials Bravelle(R): 1 vial Menopur(R)) or 300:225 IU (1 vial Bravelle(R): 3 vials of Menopur(R)) tested, the theoretical vs. actual FSH bioactivities were 150 vs. 156.86 IU/mL, 300 vs. 308.69 IU/mL and 300 vs. 306.58 IU/mL, respectively. The theoretical vs. actual LH bioactivity for Menopur(R) in the above mentioned ratios tested were 75 vs. 77.50 IU/mL. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R): 1 vial Menopur(R)), 300:75 IU (3 vials Bravelle(R): 1 vial Menopur(R)) or 300

  7. Toward Fully Synthetic Homogeneous β-Human Follicle-Stimulating Hormone (β-hFSH) with a Biantennary N-linked Dodecasaccharide. Synthesis of β-hFSH with Chitobiose Units at the Natural Linkage Sites

    PubMed Central

    Nagorny, Pavel; Fasching, Bernhard; Li, Xuechen; Chen, Gong; Aussedat, Baptiste; Danishefsky, Samuel J.

    2009-01-01

    A highly convergent synthesis of the sialic acid rich biantennary N-linked glycan found in human glycoprotein hormones, and its use in the synthesis of a fragment derived from the β-domain of human Follicle-Stimulating Hormone (hFSH) are described. The synthesis highlights the use of the Sinaÿ radical glycosidation protocol for the simultaneous installation of both biantennary side-chains of the dodecasaccharide as well as the use of glycal chemistry to construct the tetrasaccharide core in an efficient manner. The synthetic glycan was used to prepare the glycosylated 20–27aa domain of β-subunit of hFSH under a Lansbury aspartylation protocol. The proposed strategy for incorporating the prepared N-linked dodecasaccharide-containing 20–27aa domain into β-hFSH subunit was validated in the context of a model system providing, protected β-hFSH subunit functionalized with chitobiose at positions 7 and 24. PMID:19341309

  8. Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.

    PubMed

    Desai, Swapna S; Roy, Binita Sur; Mahale, Smita D

    2013-12-01

    FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.

  9. Data in support of FSH induction of IRS-2 in human granulosa cells: Mapping the transcription factor binding sites in human IRS-2 promoter.

    PubMed

    Kaur, Surleen; Anjali, G; Bhardwaj, Priya; Taneja, Jyoti; Singh, Rita

    2016-03-01

    Insulin receptor substrate-2 (IRS-2) plays critical role in the regulation of various metabolic processes by insulin and IGF-1. The defects in its expression and/or function are linked to diseases like polycystic ovary syndrome (PCOS), insulin resistance and cancer. To predict the transcription factors (TFs) responsible for the regulation of human IRS-2 gene expression, the transcription factor binding sites (TFBS) and the corresponding TFs were investigated by analysis of IRS-2 promoter sequence using MatInspector Genomatix software (Cartharius et al., 2005 [1]). The ibid data is part of author׳s publication (Anjali et al., 2015 [2]) that explains Follicle stimulating hormone (FSH) mediated IRS-2 promoter activation in human granulosa cells and its importance in the pathophysiology of PCOS. Further analysis was carried out for binary interactions of TF regulatory genes in IRS-2 network using Cytoscape software tool and R-code. In this manuscript, we describe the methodology used for the identification of TFBSs in human IRS-2 promoter region and provide details on experimental procedures, analysis method, validation of data and also the raw files. The purpose of this article is to provide the data on all TFBSs in the promoter region of human IRS-2 gene as it has the potential for prediction of the regulation of IRS-2 gene in normal or diseased cells from patients with metabolic disorders and cancer.

  10. Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

    PubMed

    Vasconcelos, G L; Saraiva, M V A; Costa, J J N; Passos, M J; Silva, A W B; Rossi, R O D S; Portela, A M L R; Duarte, A B G; Magalhães-Padilha, D M; Campelo, C C; Figueiredo, J R; van den Hurk, R; Silva, J R V

    2013-01-01

    The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 μg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

  11. Shortened estrous cycle length, increased FSH levels, FSH variance, oocyte spindle aberrations, and early declining fertility in aging senescence-accelerated mouse prone-8 (SAMP8) mice: concomitant characteristics of human midlife female reproductive aging.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Kraemer, Duane C; Morley, John E; Farr, Susan; Chaffin, Charles L; Merchenthaler, István

    2014-06-01

    Women experience a series of specific transitions in their reproductive function with age. Shortening of the menstrual cycle begins in the mid to late 30s and is regarded as the first sign of reproductive aging. Other early changes include elevation and increased variance of serum FSH levels, increased incidences of oocyte spindle aberrations and aneuploidy, and declining fertility. The goal of this study was to investigate whether the mouse strain senescence-accelerated mouse-prone-8 (SAMP8) is a suitable model for the study of these midlife reproductive aging characteristics. Midlife SAMP8 mice aged 6.5-7.85 months (midlife SAMP8) exhibited shortened estrous cycles compared with SAMP8 mice aged 2-3 months (young SAMP8, P = .0040). Midlife SAMP8 mice had high FSH levels compared with young SAMP8 mice, and mice with a single day of high FSH exhibited statistically elevated FSH throughout the cycle, ranging from 1.8- to 3.6-fold elevation on the days of proestrus, estrus, metestrus, and diestrus (P < .05). Midlife SAMP8 mice displayed more variance in FSH than young SAMP8 mice (P = .01). Midlife SAMP8 ovulated fewer oocytes (P = .0155). SAMP8 oocytes stained with fluorescently labeled antitubulin antibodies and scored in fluorescence microscopy exhibited increased incidence of meiotic spindle aberrations with age, from 2/126 (1.59%) in young SAMP8 to 38/139 (27.3%) in midlife SAMP8 (17.2-fold increase, P < .0001). Finally, SAMP8 exhibited declining fertility from 8.9 pups/litter in young SAMP8 to 3.5 pups/litter in midlife SAMP8 mice (P < .0001). The age at which these changes occur is younger than for most mouse strains, and their simultaneous occurrence within a single strain has not been described previously. We propose that SAMP8 mice are a model of midlife human female reproductive aging.

  12. Immunoreactivity of gonadotrophs (FSH and LH Cells) and gonadotropin subunit gene expression in the male chub mackerel Scomber japonicus pituitary during the reproductive cycle.

    PubMed

    Nyuji, Mitsuo; Selvaraj, Sethu; Kitano, Hajime; Shiraishi, Tetsuro; Yamaguchi, Akihiko; Shimizu, Akio; Matsuyama, Michiya

    2012-09-01

    The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique β subunit (FSHβ or LHβ); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHβ-immunoreactive (ir) and LHβ-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHβ-ir cell area increased significantly during spermiation, while FSHβ mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHβ-ir cell area and LHβ mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHβ-ir cell area and GtHβ mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHβ mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHβ-ir cell area were accompanied by similar changes in the expression of the FSHβ and LHβ genes, both of which increased during testicular development.

  13. Gene expression and secretion of LH and FSH in relation to gene expression of GnRH receptors in the brushtail possum (Trichosurus vulpecula) demonstrates highly conserved mechanisms.

    PubMed

    Crawford, J L; Heath, D A; Haydon, L J; Thomson, B P; Eckery, D C

    2009-01-01

    In eutherian mammals, the gonadotrophins (LH and FSH) are synthesized and stored in gonadotroph cells under the regulation of multiple mechanisms including GnRH. Very little is known about the regulation of gonadotrophin secretion and storage in pituitary glands of marsupials. This study revealed, using quantitative PCR and heterologous RIA techniques, that LHB mRNA expression levels remained constant over the oestrous cycle, regardless of the presence of a preovulatory LH surge, which is characteristic of a hormone secreted under regulation. Our sampling regime was unable to detect pulses of LH during the follicular phase, although GNRHR mRNA levels had increased at this time. Pulses of LH were, however, detected in the luteal phase of cycling females, in anoestrus females and in males. There was a positive correlation between gene expression of FSHB and plasma levels of FSH at different stages of the oestrous cycle and no pulses of FSH were detected at any time; all characteristics of a hormone secreted via the constitutive pathway. Using in situ hybridisation and immunohistochemistry methods, we determined that mRNA expression of LHB and FSHB, and protein storage of gonadotrophins exhibited a similar pattern of localisation within the pituitary gland. Additionally, sexual dimorphism of gonadotroph populations was evident. In summary, these findings are similar to that reported in eutherians and considering that marsupial evolution diverged from eutherians over 100 million years ago suggests that the regulation of gonadotrophins is highly conserved indeed.

  14. Regulative actions of the Chinese drugs for tonifying the kidney on gene expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP.

    PubMed

    Cai, Depei; Zhang, Wei

    2005-03-01

    It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drugs for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP, indicating that the Chinese drugs for tonifying the kidney can regulate gene expression of the hypothalamic GnRH, pituitary FSH, LH, and osteoblastic BGP, which is possibly one of the main mechanisms of the Chinese drug for tonifying the kidney, regulating ephebic development process andimproving skeletal development in sexual precocity children.

  15. Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor.

    PubMed

    Choi, Sung Sik; Jung, Joo Young; Lee, Dong Ho; Kang, Ji Yoon; Lee, Sang Ho

    2013-02-01

    Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

  16. Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

    PubMed

    Bonnet, A; Frappart, P O; Dehais, P; Tosser-Klopp, G; Hatey, F

    2006-07-07

    FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.

  17. Infertility and ovarian follicle reserve depletion are associated with dysregulation of the FSH and LH receptor density in human antral follicles.

    PubMed

    Regan, Sheena L P; Knight, Phil G; Yovich, John L; Stanger, James D; Leung, Yee; Arfuso, Frank; Dharmarajan, Arun; Almahbobi, Ghanim

    2017-02-07

    The low take-home baby rate in older women in Australia (5.8%) undergoing IVF (5.8%) is linked to the depletion of the ovarian reserve of primordial follicles. Oocyte depletion causes an irreversible change to ovarian function. We found that the young patient FSH receptor and LH receptor expression profile on the granulosa cells collected from different size follicles were similar to the expression profile reported in natural cycles in women and sheep. This was reversed in the older patients with poor ovarian reserve. The strong correlation of BMPR1B and FSH receptor density in the young was not present in the older women; whereas, the LH receptor and BMPR1B correlation was weak in the young but was strongly correlated in the older women. The reduced fertilisation and pregnancy rate was associated with a lower LH receptor density and a lack of essential down-regulation of the FSH and LH receptor. The mechanism regulating FSH and LH receptor expression appears to function independently, in vivo, from the dose of FSH gonadotrophin, rather than in response to it. Restoring an optimum receptor density may improve oocyte quality and the pregnancy rate in older women.

  18. The long-term effects of FSH and triiodothyronine administration during the pubertal period on Connexin 43 expression and spermatogenesis efficiency in adult rats.

    PubMed

    Marchlewska, Katarzyna; Slowikowska-Hilczer, Jolanta; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Filipiak, Eliza; Kula, Krzysztof

    2015-04-01

    Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Hyperstimulation of both hormones evokes regressional changes in connexin 43 expression and the seminiferous epithelium in young rats during testicular maturation. However, separate treatments with T3 reduce Sertoli cell number, which seems to be closely connected with the maturation of connexin 43 gap junctions. FSH elevates Sertoli cell number and function, but this effect may take place regardless of the presence of connexin 43-dependent intercellular communication. The aim of the study was to evaluate the later effects of such treatments. Newborn, male Wistar rats were divided randomly into experimental groups receiving daily subcutaneous injections of either 7.5 IU/animal FSH, or 100 mg/kg b.w. T3, or both substances or the same volume of vehicle (control group) until day 15 of life. The animals were sacrificed on day 50. Morphometric analysis and immunohistochemical reactions were performed using antibodies against Vimentin, Proliferating Cell Nuclear Antigen and Connexin 43 in the testis. Sertoli cell count, efficiency of spermatogenesis, and hormonal pattern were examined. Disturbances in the connexin 43 expression reduced the number of Sertoli cells, the efficiency of spermatogenesis and impaired endocrine function of testes in adult rats treated with FSH and T3 during puberty. Stimulation with FSH alone increased Sertoli cell number, but was associated with a negative effect on cell-to-cell connexin 43-dependent communication, with a consequential reduction of spermatogenesis efficiency. J. Exp. Zool. 323A: 256-265, 2015. © 2015 Wiley Periodicals, Inc.

  19. Regulation of Pcsk6 expression during the preantral to antral follicle transition in mice: opposing roles of FSH and oocytes.

    PubMed

    Diaz, Francisco J; Sugiura, Koji; Eppig, John J

    2008-01-01

    Several secreted products of the TGFbeta superfamily have important roles during follicular development and are produced by both oocytes and somatic cells (granulosa and theca) in the follicle. The proprotein convertases are a family of seven known proteins that process TGFbeta ligands and other secreted products to their mature active form. The present study examined the regulation of steady-state levels of Pcsk6 mRNA, which encodes a convertase protein known to process members of the TGFbeta superfamily, during mouse follicular development. Pcsk6 mRNA and protein were expressed in preantral but not cumulus or mural granulosa cells. Pcsk6 mRNA levels in preantral granulosa cells were not regulated by growing oocytes of preantral follicles, but were elevated by FSH. Furthermore, Pcsk6 mRNA in preantral granulosa cells was potently suppressed by factor(s) secreted by fully grown oocytes from antral follicles, in part through SMAD2/3-mediated pathways. Oocytes acquired the ability to suppress the steady-state levels of Pcsk6 mRNA in granulosa cells during the preantral to antral follicle transition. Suppression of Pcsk6 mRNA by oocytes could reflect a change in the mechanism(s) regulating the activity of members of the TGFbeta superfamily.

  20. Ovarian function and FSH receptor characteristics during canine anoestrus.

    PubMed

    McBride, M W; Aughey, E; O'Shaughnessy, P J; Jeffcoate, I A

    2001-01-01

    Ovaries of bitches are relatively inactive during anoestrus despite apparently adequate circulating FSH concentrations. Alternative FSH receptor (FSH-R) transcripts in bitches might hinder the follicular response to gonadotrophins, which may account for anoestrus. The expression of the full length FSH-R and novel isoforms in bitches was examined using in situ hybridization and RT-PCR analysis. Various PCR primers to the FSH-R were used and its expression was assessed in ovarian tissue at different stages of the oestrous cycle. RT-PCR amplification of the extracellular domain (exon 1-10) was generally successful, indicating that cFSH-R expression (> 90%) occurs throughout the oestrous cycle. Two FSH-R isoforms were sequenced, but there were no clear differences in the pattern of expression between anoestrus and other stages of the oestrous cycle, except that isoform expression was less frequent (30% occurrence) in prepubertal bitches. Data from in situ hybridization showed clear expression of the FSH-R in secondary and antral follicles, and corpora lutea. It was concluded that there is no evidence of a change in the expression of the FSH-R specific to anoestrus.

  1. [Effects of purine nucleotide on the expressions of FSH and LH and the ultrastructure of endocrine cells in the pituitary gland of heroin-addicted male rats].

    PubMed

    Cui, Jia-Yue; Hong, Xin-Yu; Wang, Shao-Hua; Liu, Jian-Kai; Cui, Li

    2012-02-01

    To investigate the effects of purine nucleotide on the expressions of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) and the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of heroin-addicted and -withdrawal rats. Ninety-two male Wistar rats were randomly divided into a control group (ip saline for 14 d), a nucleotide group (ip AMP and GMP for 10 d), a heroin group (ip heroin for 10 d), a heroin + nucleotide group (ip AMP and GMP + heroin for 10 d), a 3 d withdrawal group (ip heroin for 10 d and killed at 14 d), a 9 d withdrawal group (ip heroin for 10 d and killed at 20 d), a 3 d nucleotide group (ip nucleotide for 3 d after 10 d heroin administration and killed at 14 d), and a 9 d nucleotide group (ip nucleotide for 9 d after 10 d heroin administration and killed at 20 d). Changes in the mRNA expressions of FSH and LH in the pituitary gland of the rats were analyzed by semi-quantitative RT-PCR, and alterations in the ultrastructures of the distal somatotrophic and gonadotrophic cells were observed under the microscope. The expression of FSH mRNA was significantly increased in the nucleotide, heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.099 +/- 0.018, 0.177 +/- 0.046, 0.151 +/- 0.030 and 0.184 +/- 0.028) as compared with the control group (0.045 +/- 0.009) (P < 0.01); and so was that of LH mRNA in the heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.950 +/- 0.169, 0.990 +/- 0.171 and 0.960 +/- 0.147) in comparison with the control group (0.700 +/- 0.099) (P < 0.01). In the heroin group, the nuclei of the distal somatotrophic and gonadotrophic cells exhibited morphological abnormality, unclear membrane, slightly pyknotic matrix, marginal and agglutinated heterochromatin, dilated rough endoplasmic reticula, swollen mitochondria, broken and vacuolated cristae in the cytoplasm, obviously decreased number of secretory granules, and myelin bodies in some cells. However, the

  2. Expression of 17?-hydroxysteroid dehydrogenase and the effects of LH, FSH and prolactin on oestrone and 17?-oestradiol secretion in the endometrium of pigs during early pregnancy and the oestrous cycle.

    PubMed

    Wojciechowicz, B; Kotwica, G; Zglejc, K; Waszkiewicz, E; Franczak, A

    2016-03-07

    The endometrium of pregnant and cyclic pigs is a source of oestrone (E1) and 17β-oestradiol (E2). However, the roles of LH, FSH and prolactin (PRL) as regulators of endometrial steroidogenesis, and the presence of 17β-hydroxysteroid dehydrogenase (17β-HSD) in the porcine endometrium, remain unknown. Therefore, in the present study we examined 17β-HSD expression and the effects of LH, FSH and PRL on E1 and E2 release in vitro in endometrial explants harvested from gravid pigs on Days 10-11 (embryo migration within the uterus), 12-13 (maternal recognition of pregnancy) and 15-16 (beginning of implantation) and compared them with results obtained in non-gravid pigs. The results show that: (1) endometrial 17β-HSD activity was decreased on Days 15-16 in pregnant and cyclic pigs compared with the preceding days; (2) LH, FSH and PRL increased endometrial E1 secretion on Days 10-11 and 15-16 of pregnancy and on Days 12-13 and 15-16 of the oestrous cycle; and (3) LH, FSH and PRL increased endometrial E2 secretion on Days 15-16 of pregnancy and during the days studied in the oestrous cycle. In conclusion, data suggest that LH, FSH and PRL affect endometrial secretion of estrogens in pigs.

  3. Further evidence for direct pro-resorptive actions of FSH.

    PubMed

    Sun, Li; Zhang, Zhiyuan; Zhu, Ling-Ling; Peng, Yuanzhen; Liu, Xuan; Li, Jianhua; Agrawal, Manasi; Robinson, Lisa J; Iqbal, Jameel; Blair, Harry C; Zaidi, Mone

    2010-03-26

    We confirm that FSH stimulates osteoclast formation, function and survival to enhance bone resorption. It does so via the activation of a pertussis toxin-sensitive G(i)-coupled FSH receptor that we and others have identified on murine and human osteoclast precursors and mature osteoclasts. FSH additionally enhances the production of several osteoclastogenic cytokines, importantly TNFalpha, likely within the bone marrow microenvironment, to augment its pro-resorptive action. FSH levels in humans rise before estrogen falls, and this hormonal change coincides with the most rapid rates of bone loss. On the basis of accumulating evidence, we reaffirm that FSH contributes to the rapid peri-menopausal and early post-menopausal bone loss, which might thus be amenable to FSH blockade.

  4. The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

    PubMed

    Chen, Mei-Jou; Chou, Chia-Hung; Chen, Shee-Uan; Yang, Wei-Shiung; Yang, Yu-Shih; Ho, Hong-Nerng

    2015-12-17

    Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.

  5. The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

    PubMed Central

    Chen, Mei-Jou; Chou, Chia-Hung; Chen, Shee-Uan; Yang, Wei-Shiung; Yang, Yu-Shih; Ho, Hong-Nerng

    2015-01-01

    Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells. PMID:26674985

  6. Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate.

    PubMed

    McNatty, K P; Lun, S; Heath, D A; Hudson, N L; O'Keeffe, L E; Henderson, K M

    1989-05-01

    At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.

  7. GnRH-agonist implantation of prepubertal male cats affects their reproductive performance and testicular LH receptor and FSH receptor expression.

    PubMed

    Mehl, N S; Khalid, M; Srisuwatanasagul, S; Swangchan-Uthai, T; Sirivaidyapong, S

    2016-03-15

    This study was conducted to investigate the effect of GnRH-agonist implantation in prepubertal tomcats on sexual behavior, reproductive performance, and expression of testicular LH receptor (LHR) and FSH receptor (FSHR) and also to compare the testicular characteristics, LHR and FSHR expression between prepubertal and adult tomcats. In experiment 1, 3-month-old tomcats (n = 6/group) were either treated with or left without 4.7 mg deslorelin implants. Semen collection and evaluation were performed just before castration at 48 weeks after treatment; removed testes were analyzed for mRNA and protein expression of LHR and FSHR. We were able to collect semen from six non-treated cats, whereas in treated cats, semen was uncollectable. The results revealed that sexual behavior was absent in the implanted cats throughout the study period. Testicular volume was found to decrease from 30 weeks after treatment onward in the implanted cats compared to the controls (P < 0.05). Semen production was found only in non-implanted cats. Testicular tissue score, seminiferous tubule diameter, and LHR protein expression were found lower in the implanted cats (P < 0.05), but no differences were observed in mRNA expression of LHR and protein expression of FSHR between groups. The mRNA expression of FSHR was higher in the implanted (P < 0.05) compared to control cats. In experiment 2, testes from prepubertal (n = 6) and adult (n = 6) male cats were collected after castration and analyzed for mRNA and protein expression of LHR and FSHR. No differences were observed in the protein expression of LHR and FSHR between the two groups, whereas mRNA expression of FSHR was higher in prepubertal cats (P < 0.05). Testicular and epididymal weight, diameter of seminiferous tubules, and the testicular grade were higher in the adult compared to prepubertal cats (P < 0.05). In conclusion, deslorelin implants suppressed protein expression of LHR and enhanced mRNA expression of FSHR along with suppression

  8. Type of gonadotropin used during controlled ovarian stimulation induces differential gene expression in human cumulus cells: A randomized study.

    PubMed

    Cruz, María; Requena, Antonio; Agudo, David; García-Velasco, Juan Antonio

    2017-08-01

    The cumulus-oocyte complex plays a central role in the regulation of folliculogenesis where it is important for the maturation, reprogramming, and fertilization of oocytes. Consequently, cumulus cell gene expression profiling is being explored as a promising method for assessing oocyte competence in the near future. Through DNA microarray technology, we analyzed the potential differences in the gene expression profiles of cumulus cells from preovulatory follicles after controlled ovarian stimulation using different types of gonadotropins. A prospective, randomized study was performed among 90 women participating in an oocyte donation program. Subjects were assigned to receive recombinant follicle-stimulating hormone (FSH), urinary FSH, or human menopausal gonadotropin (hMG). The gene expression profile in cumulus cells was analyzed according the type of gonadotropin received during ovarian stimulation. Furthermore, we also performed a gene ontology analysis to provide structural knowledge. Hierarchical clustering, principal component analysis, and gene enrichment analysis revealed greater differences between the urinary FSH and hMG groups compared to the rest of the pair-wise comparisons; recombinant FSH vs hMG and urinary FSH vs recombinant FSH. Data suggest that controlled ovarian stimulation induces specific gene expression profiles in human cumulus cells depending on the type of gonadotropin used. Registered at clinicaltrials.gov; identifier NCT022437032. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Revisiting the expression and function of follicle-stimulation hormone receptor in human umbilical vein endothelial cells

    PubMed Central

    Stelmaszewska, Joanna; Chrusciel, Marcin; Doroszko, Milena; Akerfelt, Malin; Ponikwicka-Tyszko, Donata; Nees, Matthias; Frentsch, Marco; Li, Xiangdong; Kero, Jukka; Huhtaniemi, Ilpo; Wolczynski, Slawomir; Rahman, Nafis A.

    2016-01-01

    Expression of follicle-stimulation hormone receptor (FSHR) is confined to gonads and at low levels to some extragonadal tissues like human umbilical vein endothelial cells (HUVEC). FSH-FSHR signaling was shown to promote HUVEC angiogenesis and thereafter suggested to have an influential role in pregnancy. We revisited hereby the expression and functionality of FSHR in HUVECs angiogenesis, and were unable to reproduce the FSHR expression in human umbilical cord, HUVECs or immortalized HUVECs (HUV-ST). Positive controls as granulosa cells and HEK293 cells stably transfected with human FSHR cDNA expressed FSHR signal. In contrast to positive control VEGF, FSH treatment showed no effects on tube formation, nitric oxide production, wound healing or cell proliferation in HUVEC/HUV-ST. Thus, it remains open whether the FSH-FSHR activation has a direct regulatory role in the angiogenesis of HUVECs. PMID:27848975

  10. Stimulatory effect of a specific substance P antagonist (RPR 100893) of the human NK1 receptor on the estradiol-induced LH and FSH surges in the ovariectomized cynomolgus monkey.

    PubMed

    Kerdelhué, B; Gordon, K; Williams, R; Lenoir, V; Fardin, V; Chevalier, P; Garret, C; Duval, P; Kolm, P; Hodgen, G; Jones, H; Jones, G S

    1997-10-01

    Utilizing a human NK1 receptor antagonist (RPR 100893), the present in vivo study was designed to test the hypothesis that endogenous substance P (SP) modulates the action of 17beta-estradiol in inducing luteinizing hormone (LH) and follicle stimulating hormone (FSH) surges in ovariectomized cynomolgus monkey. Plasma concentrations of LH and FSH as well as NK1 receptor antagonist and SP were measured during the development of the negative and positive feedback phases which follow a single administration of estradiol benzoate (50 microg/kg) to long-term ovariectomized monkeys. Daily administration by gastric intubation of 1 mg/kg or 10 mg/kg of the NK1 receptor antagonist (RPR 100893) leads to detectable levels of the antagonist in the blood of treated animals for at least 6 hr after its administration. These levels are in agreement with the experimentally determined IC50 value of the antagonist. The most striking finding of this study is that LH and FSH releases are enhanced during the descending arm of the estradiol benzoate-induced LH and FSH surges, which suggests that endogenous SP normally has an inhibitory role during this time. The enhancement of LH release is approximately 50%, regardless of the amount of the NK1 antagonist used. However, the enhanced FSH release is more important. Furthermore, blockade of the NK1 receptor with the smaller dose of the antagonist leads to a small, but significant, increase in plasma levels of SP, indicating that blockade of SP receptors leads to an increased release of SP. Collectively, these results further substantiate the link which exists between the ovarian steroid 17beta-estradiol and SP systems. Also, for the first time, these results demonstrate an inhibitory involvement of the human NK1 receptor in the 17beta-estradiol-induced pseudo-ovulatory gonadotropin surges in the ovariectomized monkey.

  11. Regulation of expression of Sertoli cell glucose transporters 1 and 3 by FSH, IL1 beta, and bFGF at two different time-points in pubertal development.

    PubMed

    Galardo, María Noel; Riera, María Fernanda; Pellizzari, Eliana Herminia; Chemes, Héctor Edgardo; Venara, Marcela Cristina; Cigorraga, Selva Beatriz; Meroni, Silvina Beatriz

    2008-11-01

    Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.

  12. Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

    PubMed

    Chauvigné, François; Verdura, Sara; Mazón, María José; Boj, Mónica; Zanuy, Silvia; Gómez, Ana; Cerdà, Joan

    2015-09-15

    In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

  13. Macroorchidism and panhypopituitarism: two different forms of presentation of FSH-secreting pituitary adenomas in adolescence.

    PubMed

    Clemente, María; Caracseghi, Fabiola; Gussinyer, Miquel; Yeste, Diego; Albisu, Marian; Vázquez, Elida; Ortega, Arantxa; Carrascosa, Antonio

    2011-01-01

    FSH-secreting pituitary adenomas are extremely rare in children and are seldom associated with clinical manifestations of high serum gonadotrophin levels. Thus, most patients have a late presentation, usually as macroadenomas. Two different clinical forms of presentation of FSH-secreting pituitary adenomas are reported: one in a 12-year-old boy with macroorchidism due to a pituitary microadenoma, probably FSH-secreting, and the other in a 15-year-old boy with panhypopituitarism due to an FSH-producing macroadenoma. Both patients presented slightly high or high FSH with low LH and high inhibin B levels. In the first case, the microadenoma was treated medically with cabergoline, which failed to reduce FSH and inhibin B levels. No radiological progression has been observed despite increasing testicular volume. In the second case, surgery was performed on the macroadenoma, leading to a decrease in FSH and inhibin B levels. The patient developed severe hypothalamic obesity and is currently under treatment with somatostatin. FSH-secreting pituitary tumors have an extremely variable clinical expression. The discrepancy between normal or slightly increased FSH and low LH values, together with high inhibin B levels, strongly suggests FSH hypersecretion which should be studied. Copyright © 2010 S. Karger AG, Basel.

  14. FSH (Follicle-Stimulating Hormone) Test

    MedlinePlus

    ... follicle-stimulating hormone (FSH), a hormone associated with reproduction and the development of eggs in women and ... FSH and LH with the development of secondary sexual characteristics at an unusually young age are an ...

  15. Pituitary gonadotropins FSH and LH are oppositely regulated by the activin/follistatin system in a basal teleost, the eel.

    PubMed

    Aroua, Salima; Maugars, Gersende; Jeng, Shan-Ru; Chang, Ching-Fong; Weltzien, Finn-Arne; Rousseau, Karine; Dufour, Sylvie

    2012-01-01

    European eels are blocked at a prepubertal silver stage due to a deficient production of pituitary gonadotropins. We investigated the potential role of activin/follistatin system in the control of eel gonadotropins. Through the development of qPCR assays for European eel activin β(B) and follistatin, we first analyzed the tissue distribution of the expression of these two genes. Both activin β(B) and follistatin are expressed in the brain, pituitary and gonads. In addition, a striking expression of both transcripts was also found in the retina and in adipose tissue. The effects of recombinant human activins and follistatin on eel gonadotropin gene expression were studied using primary cultures of eel pituitary cells. Activins A and B strongly stimulated FSHβ subunit expression in a time- and dose-dependent manner. In contrast, activin reduced LHβ expression, an inhibitory effect which was highlighted in the presence of testosterone, a known activator of eel LHβ expression. No effect of activin was observed on other pituitary hormones. Follistatin antagonized both the stimulatory and inhibitory effects of activin on FSHβ and LHβ expression, respectively. Activin is the first major stimulator of FSH expression evidenced in the eel. These results in a basal teleost further support the ancient origin and strong conservation of the activin/follistatin system in the control of FSH in vertebrates. In contrast, the opposite regulation of FSH and LH may have emerged in the teleost lineage.

  16. Gene expression analysis of bovine oocytes at optimal coasting time combined with GnRH antagonist during the no-FSH period.

    PubMed

    Labrecque, Rémi; Vigneault, Christian; Blondin, Patrick; Sirard, Marc-André

    2014-05-01

    Ovarian stimulation with FSH combined with an appropriate period of FSH withdrawal (coasting) before ovum pick-up now appears to be a successful way to obtain oocytes with high developmental competence in bovine. Recent results showed that extending follicular growth by only 24 hours has a detrimental effect on oocyte quality as shown by the reduced blastocyst formation rate. Although these treatments are initiated during the luteal phase with low LH level, the small LH pulsatility present at that time could potentially impact follicular development as well as oocyte quality. In this study, a GnRH antagonist (Cetrotide) was used to suppress LH secretion during follicular differentiation to get a better insight into the physiological importance of the LH support during that period. Oocytes were collected by ovum pick-up, and quality was assessed by measuring the blastocyst formation rate obtained after IVM-IVF. The oocyte transcriptome from GnRH antagonist-treated animals was also compared with that from a control group (coasting duration of 68 hours) to detect possible alterations at the messenger RNA (mRNA) level. The oocyte quality was not statistically affected by the treatment as shown by the blastocyst formation rate obtained. However, microarray analysis showed that a total of 226 genes had a significant difference (fold change > 2; P < 0.05) at the mRNA level, with the majority being in overabundance in the treated group. Many genes related to RNA posttranscriptional modifications presented different abundance at the mRNA level significant differences in the control group (68 hours), whereas translation function appeared to be affected, with many genes related to structural constituents of the ribosome presenting a overabundance in the GnRH antagonist-treated group. Specific mRNAs with crucial roles in chromosome segregation control also showed significant difference at the mRNA level after Cetrotide treatment. The results presented here indicated that the

  17. Hormonal treatment of male infertility: FSH.

    PubMed

    Foresta, C; Selice, R; Ferlin, A; Arslan, P; Garolla, A

    2007-12-01

    FSH plays a crucial role in spermatogenesis. In the fetal and neonatal development stages, FSH activates the proliferation of the Sertoli cells and successively, during the pubertal phase, it influences the mitotic activity of the spermatogonia and encourages cellular differentiation, until arrival at the round spermatid stage. Because of its physiological role in spermatogenesis, various attempts have been made to treat idiopathic oligozoospermic men with FSH. However, the results obtained so far are still controversial. In this research, attention was focused on the possible criteria able to predict a seminal response to this specific hormonal treatment. Furthermore, the effectiveness of FSH therapy was evaluated in terms of sperm count and pregnancy rate. Thus far, based on more recent knowledge about the FSH receptor gene, the authors have correlated different polymorphisms of this gene with the outcome of FSH treatment. In this paper, the literature is reviewed and the authors' experience on using FSH in male infertility is discussed.

  18. [From theory to clinical practice: recombinant FSH in daily practice].

    PubMed

    Kably Ambe, A; Barrón Vallejo, J; Góngora Rodríguez, A; Carballo Mondragón, E; Anta Jaén, E

    1999-08-01

    The purpose of the present study is to determine the efficacy of induction ovulation with recombinant FSH in patients treated with in vitro fertilization and embryo transfer (IVF-ET) and basic assisted reproductive techniques (ART). One hundred seven cycles were analyzed. The patients were divided in two groups: Group 1, treated with IVF (n = 12) and group 2, treated with basic ART (n = 95). Only recombinant FSH was utilized for ovulation induction; human corionic gonadotropin (hCG), 10,000 IU, were administered when one or more dominant follicles with diameter > or = 18 mm were presents; oocyte retrieval was performed 34 hour, while intrauterine insemination was practiced at 36 hours after the hCG injection. The pregnancy rate per IVF cycle was 25.0%, and 16.4% for basic ART. It is concluded that ovulation induction with recombinant FSH is a good and efficient alternative for both variations of ART.

  19. LAPS-FSH: a new and effective long-acting follicle-stimulating hormone analogue for the treatment of infertility.

    PubMed

    Jung, Sunyoung; Park, Youngjin; Kim, YoungHoon; Kim, Yu Yon; Choi, Hyun-Ji; Son, Woo-Chan; Kwon, SeChang

    2014-10-01

    Although several long-acting follicle-stimulating hormone (FSH) therapies have been developed to enhance the ovarian response, a disadvantage of FSH therapy is its relatively short half-life, which requires women to receive one to two injections per day for almost 2 weeks. In the present study, we developed a novel FSH analogue by conjugating recombinant human FSH (rhFSH) and the constant region of the human immunoglobulin G4 fragment via non-peptidyl linkers. The efficacy of the FSH analogue was evaluated in vitro by cAMP level assessments, pharmacokinetic studies and a determination of ovarian weight and by comparing these findings with the results from other FSH analogues. In addition, the total number of antral and Graafian follicles was determined after 7 days of treatment with control, 6µgkg(-1) follitropin β, 6, 12 or 42µgkg(-1) corifollitropin α or 3, 6 or 12µgkg(-1) long acting protein/peptide discovery-follicle-stimulating hormone (LAPS-FSH). As a result, the animals treated with 12µgkg(-1) LAPS-FSH produced additional and larger healthy follicles. These data demonstrate that LAPS-FSH promotes growth and inhibits atresia of the ovarian follicle compared with other available drugs, suggesting that our new drug enhances the efficacy and duration of treatment. It is expected that our new FSH analogue will result in a higher chance of pregnancy in patients who are unresponsive to other drugs.

  20. Neuroendocrine function in adult female transgenic mice expressing the human growth hormone gene.

    PubMed

    Chandrashekar, V; Bartke, A; Wagner, T E

    1992-04-01

    Adult female transgenic mice expressing the human GH (hGH) gene with mouse metallothionein-I promoter are sterile. To evaluate the hypothalamic-pituitary function in these animals, adult female transgenic mice and nontransgenic normal littermates were ovariectomized. On days 7 and 8 after ovariectomy, mice were injected with either oil or primed with 0.5 micrograms estradiol benzoate (EB) in oil, 24 h later treated with 10 micrograms EB/100 g body wt and a day later bled for measurements of FSH, LH, and PRL levels. Plasma gonadotropin and PRL levels were also measured in ovary-intact transgenic and normal siblings at estrus. Additional ovariectomized EB-treated transgenic mice and normal siblings were injected with either saline or GnRH in saline (1 ng/g body wt) and were bled 15 min later for determination of circulating hormone levels. At estrus, in transgenic mice, circulating FSH and PRL levels were significantly lower (FSH:P less than 0.001; PRL:P less than 0.025), but plasma LH concentrations were higher (P less than 0.001) than those in nontransgenic mice. As expected, ovariectomy significantly increased (P less than 0.001) circulating FSH and LH levels in both groups of mice relative to ovary-intact animals, but the increase in plasma LH levels was attenuated in transgenic mice. The suppressive effect of estrogen on circulating FSH and LH levels were similar in transgenic and nontransgenic mice. Treatment with GnRH significantly increased plasma FSH and LH levels in both transgenic and normal mice. However, the plasma FSH and LH responses to GnRH administration were significantly reduced (P less than 0.001) in transgenic mice. The results of these studies indicate that adult female transgenic mice expressing the hGH gene are hypoprolactinemic. Yet due to PRL-like activity of hGH, the gonadotropin secretion is altered. Thus, endogenously secreted hGH modulates the hypothalamic-pituitary function of adult female transgenic mice bearing the hGH gene.

  1. Unexpected effect of the vehicle (grain ethanol) of homeopathic FSH on the in vitro survival and development of isolated ovine preantral follicles.

    PubMed

    Lima, Lartiza F; Rocha, Rebeca M P; Duarte, Ana Beatriz G; Brito, Ivina R; Silva, Gerlane M; Rodrigues, Giovanna Q; Nunes-Pinheiro, Diana C S; Sales, Antônia D; Moura, Arlindo A; Wheeler, Matthew B; Rodrigues, Ana Paula R; Campello, Cláudio C; Figueiredo, José Ricardo

    2017-04-01

    The aims of this study were to investigate the effects of medium replacement system (experiment I) and of FSH presentations (homeopathic - FSH 6cH and allopathic FSH - rFSH; experiment II) on the in vitro development, hormone production and gene expression of isolated ovine preantral follicles cultured for 6 days. In experiment I, secondary follicles were cultured in the α-MEM(+) supplemented with FSH 6cH (0.05 fg/ml) or recombinant bovine FSH (100 ng/ml) without/with daily medium addition. The homeopathic FSH treatments with/without medium addition improved (p < .05) follicular development compared to rFSH100 treatment without addition. FSH 6cH with addition showed the highest (p < .05) estradiol production. To verify whether the effects of homeopathic FSH were not due to its vehicle, experiment II was performed. The α-MEM(+) was supplemented or not with alcohol (0.2% grain ethanol, v/v), FSH 6cH or rFSH100 with daily medium addition. Surprisingly, we found that all treatments improved follicular development compared to the α-MEM(+) (p < .05). Moreover, homeopathic FSH was similar to the other treatments including its vehicle. In conclusion, its vehicle (ethanol) causes the effect of homeopathic FSH on in vitro development of isolated ovine preantral follicles.

  2. Proresorptive actions of FSH and bone loss.

    PubMed

    Zaidi, Mone; Blair, Harry C; Iqbal, Jameel; Zhu, Ling Ling; Kumar, T Rajendra; Zallone, Alberta; Sun, Li

    2007-11-01

    We review studies that propose follicle-stimulating hormone (FSH) as a physiologic stimulator of osteoclastic bone resorption. We hypothesize that, in addition to low estrogen, a rising FSH contributes to the increased bone resorption and bone loss in hypergonadism. This is of particular relevance to the perimenopausal transition, wherein profound bone loss is accompanied by trabecular perforations in the face of high FSH and normal estrogen levels. Potential therapeutic implications include the development of antagonists to both circulating FSH and its osteoclastic receptor.

  3. mRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

    PubMed Central

    Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick

    2012-01-01

    FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m7GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5′cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

  4. Characterization of FSH signalling networks in bovine cumulus cells: a perspective on oocyte competence acquisition.

    PubMed

    Khan, D R; Guillemette, C; Sirard, M A; Richard, F J

    2015-09-01

    Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.

  5. Identifying real differences in live birth rates between HMG and rFSH in IVF.

    PubMed

    Afnan, Masoud

    2009-01-01

    Fertility treatment strives for the delivery of a healthy live birth. Human menopausal gonadotrophin (HMG) and recombinant FSH (rFSH) are the two types of gonadotrophin currently used for ovarian stimulation in assisted reproduction treatments. Although both HMG and rFSH have been shown to be effective, a number of studies have examined whether a potential difference in clinical benefit or outcome exists between treatments. Unlike rFSH preparations, HMG contains both FSH and LH activity (in the form of LH and human chorionic gonadotrophin, which are short- and long-acting, respectively). The beneficial effect of exogenous LH activity has been investigated in the Menotrophin versus Recombinant FSH in-vitro Fertilisation Trial (MERiT), which revealed differences in embryo quality and endometrial receptivity between rFSH and highly purified HMG. Current evidence suggests that HMG provides significantly higher live birth rates than rFSH in women undergoing ovarian stimulation for in-vitro fertilization/intracytoplasmic sperm injection cycles using long gonadotrophin-releasing hormone agonist protocol. Further studies will continue to provide data with which to expand these findings and optimize the chances of achieving a live birth following assisted reproduction treatment.

  6. Molecular pathology of the FSH receptor: new insights into FSH physiology.

    PubMed

    Meduri, G; Bachelot, A; Cocca, M P; Vasseur, C; Rodien, P; Kuttenn, F; Touraine, P; Misrahi, M

    2008-01-30

    Manipulations of mouse genome have helped to elucidate gonadotrophin function but important differences subsist between rodent and human reproduction. Studies of patients with mutations of gonadotrophins or gonadotrophin receptors genes allow understanding their physiological effects in humans. The correlation of the clinical phenotypes of patients with in vitro studies of the mutated receptor residual function and histological and immunohistological studies of the ovarian biopsies permits to understand which stages of follicular development are under FSH control. Total FSH receptor (FSHR) inactivation causes infertility with an early block of follicular maturation remarkably associated with abundant small follicles as in prepubertal ovaries and demonstrates the absolute requirement of FSH for follicular development starting from the primary stage. Partial FSHR inactivation, characterized by normal-sized ovaries, can sustain follicular development up to the early antral stages but incremental levels of FSH stimulation seem to be required for antral follicular growth before selection. These findings contrast with the traditional view of an initial gonadotrophin-independent follicular growth prior to the preantral-early antral stages. The presence of numerous reserve follicles in the ovaries of these patients may permit a future treatment of their infertility. The study of reduced FSHbeta or FSHR activity in genetically modified male mice models and in men suggests a minor impact of the FSHR on masculine fertility. Further studies on patients with a demonstrated total FSHbeta or FSHR inactivation are required to elucidate reported differences in spermatogenesis impairment. Finally, the studies of mutations of gonadotrophins and their receptors demonstrate differences in gonadotrophin function between genetically modified rodents and humans which suggest prudence in extrapolating observations in rodents to human reproduction. Ovarian hyperstimulation syndrome (OHSS

  7. Sequential Versus Continual Purified Urinary FSH/hCG in Men With Idiopathic Hypogonadotropic Hypogonadism.

    PubMed

    Zhang, Manna; Tong, Guoyu; Liu, Yanling; Mu, Yiming; Weng, Jianping; Xue, Yaoming; Luo, Zuojie; Xue, Yuanming; Shi, Lixin; Wu, Xueyan; Sun, Shouyue; Zhu, Yanhua; Cao, Ying; Zhang, Jie; Huang, Hong; Niu, Ben; Li, Hong; Guo, Qinghua; Gao, Yan; Li, Zhibin; Ning, Guang; Zhu, Dalong; Li, Xiaoying

    2015-06-01

    Gonadotropin therapy using a human chorionic gonadotropin (hCG) and FSH preparation is an effective regimen in inducing masculinization and spermatogenesis in men with idiopathic hypogonadotropic hypogonadism (IHH). However, the high cost of medication and frequent injections affect compliance. The aim of this study was to determine the efficacy of sequential use of highly purified urinary FSH (uFSH)/hCG in men with IHH. A randomized, open-label, prospective, controlled noninferiority trial with an 18-month follow-up was conducted in 9 tertiary hospitals. A total of 67 Chinese men with IHH were randomly allocated into group A receiving continual uFSH (75 U, 3 times a week) and hCG (2000 U, twice a week) injection and group B receiving sequential uFSH (75 U, 3 times a week every other 3 months) and hCG (2000 U, twice a week) injection. The primary outcome was the proportion of subjects with a sperm concentration of ≥ 1.0 × 10(6)/mL during the 18 months. The efficacy between groups A and B was compared for noninferiority. Of the patients, 17/33 (51.5%) receiving continual uFSH/hCG and 19/34 (55.9%) receiving sequential uFSH/hCG achieved sperm concentrations of ≥ 1.0 × 10(6)/mL. The efficacy in the sequential uFSH/hCG group was not inferior to that in the continual uFSH/hCG group (noninferiority, P = .008) by intention-to-treat analysis. The efficacy of the sequential uFSH/hCG regimen is not inferior to that of the continual uFSH/hCG regimen in inducing spermatogenesis and masculinization of patients with IHH.

  8. Enhancement of FSH bioactivity in vivo using site-specific antisera.

    PubMed

    Ferasin, L; Gabai, G; Beattie, J; Bono, G; Holder, A T

    1997-03-01

    ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species.

  9. FSH and bFGF stimulate the production of glutathione in cultured rat Sertoli cells.

    PubMed

    Gualtieri, Ariel F; Mazzone, Graciela L; Rey, Rodolfo A; Schteingart, Helena F

    2009-06-01

    Migration of developing germ cells from the basal to the adluminal compartment of the seminiferous epithelium requires extensive tissue restructuring, resulting in the production of reactive oxygen species. Sertoli cells are involved in this process. Glutathione (GSH), produced by Sertoli cells, has an essential role in cell protection against oxidative stress. Intracellular GSH content is maintained by de novo synthesis, involving glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, and by recycling from oxidized GSH, catalysed by glutathione reductase (GR). To assess whether follicle-stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) modulate GSH production in Sertoli cells by regulating the expression of GCLC, GCLM and/or GR, we performed in vitro studies using rat Sertoli cells in primary culture. FSH and bFGF stimulation increased Sertoli cell GSH levels after 24 h incubation. The simultaneous addition of FSH and bFGF did not produce any further effect. GCLM expression was upregulated by FSH and bFGF 6 h. At 24 h, only the FSH-mediated effect was still observed. FSH and bFGF also upregulated GR expression. In conclusion, our results show that FSH and bFGF increase GSH levels in Sertoli cells through stimulation of the de novo synthesis and recycling by upregulating GCLM and GR expression respectively. Therefore, protection of germ cells against oxidative stress seems to be regulated by hormones and germ cell-released growth factors capable of influencing the production of Sertoli cell GSH.

  10. [Ovulation induction with pure FSH in anovulatory patients resistant to clomifene citrate].

    PubMed

    Kably-Ambe, A; Reyes-Cuervo, H; Barrón-Vallejo, J

    1996-07-01

    The objective was to evaluate the utility of the pure FSH as treatment of women clomiphene-resistant. Seventy two patients clomiphene-resistant were treated with pure FSH. Ovulation induction was started with 75 IU of pure FSH on day 3 of the menstrual cycle, monitoring the follicular growth with transvaginal ultrasonography, additional doses of pure FSH were administered accordingly. Human chorionic gonadotropin (10,000 IU) was administered when the dominant follicle reached a diameter > or = 16 mm. The pregnancy rate per cycle was 18.0%, on the other hand, the cumulate rate of pregnancy was 72.2%. There was not significant difference in the pregnancy rate between patients with primary or secondary infertility. The rate of spontaneous abortions was similar to the general population. As conclusion, it therefore appropriate to offer the treatment with pure FSH to patients clomiphene-resistance. The cases with gonadotropin-resistance, will be candidates to surgical procedures.

  11. Dose of recombinant FSH and oestradiol concentration on day of HCG affect embryo development kinetics.

    PubMed

    Muñoz, Manuel; Cruz, María; Humaidan, Peter; Garrido, Nicolás; Pérez-Cano, Inmaculada; Meseguer, Marcos

    2012-10-01

    During follicular growth, the follicle is exposed to an almost ever-changing composition of isoforms of FSH and LH, which causes a number of different and divergent biological effects. Through a time-lapse system, embryo kinetics were examined following the use of FSH only (recombinant FSH, rFSH) and gonadotrophins containing LH activity (human menopausal gonadotrophin, HMG, and FSH+HMG) in oocyte donors. No significant differences were seen between the three groups (for rFSH, HMG and rFSH+HMG, t2 was 27.8h, 27.9h and 27.5h respectively). Moreover, although embryos obtained with rFSH showed an increase in the proportions of optimal timings of development, the differences observed were not significant, as shown by the percentages of embryos inside/outside these kinetic variables. In contrast, for gonadotrophin dosage and oestradiol concentration, this study observed differences in embryo development kinetics for some of the variables evaluated, which allowed the description of an optimal range of gonadotrophin dosage and oestradiol concentration. However, these kinetic differences did not translate into important distinctions in the proportion of optimal embryos with a higher implantation potential.

  12. Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure.

    PubMed

    Bousfield, George R; Butnev, Vladimir Y; Bidart, Jean-Michel; Dalpathado, Dilusha; Irungu, Janet; Desaire, Heather

    2008-02-12

    Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the

  13. Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

    PubMed

    Andreone, Luz; Ambao, Veronica; Pellizzari, Eliana Herminia; Loreti, Nazareth; Cigorraga, Selva; Campo, Stella

    2017-08-30

    Variations in FSH carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the Sertoli cell. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (InhB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated InhB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated InhB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and InhB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased InhB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on InhB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated InhB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

  14. Cooperative Effects of FOXL2 with the Members of TGF-β Superfamily on FSH Receptor mRNA Expression and Granulosa Cell Proliferation from Hen Prehierarchical Follicles.

    PubMed

    Qin, Ning; Fan, Xian-Cong; Xu, Xiao-Xing; Tyasi, Thobela Louis; Li, Shi-Jun; Zhang, Ying-Ying; Wei, Man-Li; Xu, Ri-Fu

    2015-01-01

    Forkhead box L2 (FOXL2) is a member of the forkhead nuclear factor 3 gene family and plays an essential role in ovarian growth and maturation in mammals. However, its potential effects and regulative mechanism in development of chicken ovarian prehierarchical follicles remain unexplored. In this study, the cooperative effects of FOXL2 with activin A, growth differentiation factor-9 (GDF9) and follistatin, three members of the transforming growth factor beta (TGF-β) superfamily that were previously suggested to exert a critical role in follicle development was investigated. We demonstrated herein, using in-situ hybridization, Northern blot and immunohistochemical analyses of oocytes and granulosa cells in various sizes of prehierarchical follicles that both FOXL2 transcripts and FOXL2 proteins are predominantly expressed in a highly similar expression pattern to that of GDF9 gene. In addition, the FOXL2 transcript was found at lower levels in theca cells in the absence of GDF9. Furthermore, culture of granulosa cells (GCs) from the prehierarchical follicles (6-8 mm) in conditioned medium revealed that in the pcDNA3.0-FOXL2 transfected GCs, there was a more dramatic increase in FSHR mRNA expression after treatment with activin A (10 ng/ml) or GDF9 (100 ng/ml) for 24 h which caused a stimulatory effect on the GC proliferation. In contrast, a significant decrease of FSHR mRNA was detected after treatment with follistatin (50 ng/ml) and resulted in an inhibitory effect on the cell proliferation. The results of this suggested that FOXL2 plays a bidirectional modulating role involved in the intracellular FSHR transcription and GC proliferation via an autocrine regulatory mechanism in a positive or negative manner through cooperation with activin A and/or GDF9, and follistatin in the hen follicle development. This cooperative action may be mediated by the examined Smad signals and simultaneously implicated in modulation of the StAR, CCND2, and CYP11A1 expression.

  15. Cooperative Effects of FOXL2 with the Members of TGF-β Superfamily on FSH Receptor mRNA Expression and Granulosa Cell Proliferation from Hen Prehierarchical Follicles

    PubMed Central

    Qin, Ning; Fan, Xian-Cong; Xu, Xiao-Xing; Tyasi, Thobela Louis; Li, Shi-Jun; Zhang, Ying-Ying; Wei, Man-Li; Xu, Ri-Fu

    2015-01-01

    Forkhead box L2 (FOXL2) is a member of the forkhead nuclear factor 3 gene family and plays an essential role in ovarian growth and maturation in mammals. However, its potential effects and regulative mechanism in development of chicken ovarian prehierarchical follicles remain unexplored. In this study, the cooperative effects of FOXL2 with activin A, growth differentiation factor-9 (GDF9) and follistatin, three members of the transforming growth factor beta (TGF-β) superfamily that were previously suggested to exert a critical role in follicle development was investigated. We demonstrated herein, using in-situ hybridization, Northern blot and immunohistochemical analyses of oocytes and granulosa cells in various sizes of prehierarchical follicles that both FOXL2 transcripts and FOXL2 proteins are predominantly expressed in a highly similar expression pattern to that of GDF9 gene. In addition, the FOXL2 transcript was found at lower levels in theca cells in the absence of GDF9. Furthermore, culture of granulosa cells (GCs) from the prehierarchical follicles (6–8 mm) in conditioned medium revealed that in the pcDNA3.0-FOXL2 transfected GCs, there was a more dramatic increase in FSHR mRNA expression after treatment with activin A (10 ng/ml) or GDF9 (100 ng/ml) for 24 h which caused a stimulatory effect on the GC proliferation. In contrast, a significant decrease of FSHR mRNA was detected after treatment with follistatin (50 ng/ml) and resulted in an inhibitory effect on the cell proliferation. The results of this suggested that FOXL2 plays a bidirectional modulating role involved in the intracellular FSHR transcription and GC proliferation via an autocrine regulatory mechanism in a positive or negative manner through cooperation with activin A and/or GDF9, and follistatin in the hen follicle development. This cooperative action may be mediated by the examined Smad signals and simultaneously implicated in modulation of the StAR, CCND2, and CYP11A1 expression. PMID

  16. Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

    PubMed

    Sicinski, P; Donaher, J L; Geng, Y; Parker, S B; Gardner, H; Park, M Y; Robker, R L; Richards, J S; McGinnis, L K; Biggers, J D; Eppig, J J; Bronson, R T; Elledge, S J; Weinberg, R A

    1996-12-05

    THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

  17. Production of biologically active recombinant goose FSH in a single chain form with a CTP linker sequence.

    PubMed

    Li, Hui; Zhu, Huanxi; Qin, Qinming; Lei, Mingming; Shi, Zhendan

    2017-02-01

    FSH is a glycoprotein hormone secreted by the pituitary gland that is essential for gonadal development and reproductive function. In avian reproduction study, especially in avian reproduction hormone study, it is hindered by the lack of biologically active FSH. In order to overcome this shortcoming, we prepared recombinant goose FSH as a single chain molecule and tested its biological activities in the present study. Coding sequences for mature peptides of goose FSH α and β subunits were amplified from goose pituitary cDNA. A chimeric gene containing α and β subunit sequences linked by the hCG carboxyl terminal peptide coding sequence was constructed. The recombinant gene was inserted into the pcDNA3.1-Fc eukaryotic expression vector to form pcDNA-Fc-gFSHβ-CTP-α and then transfected into 293-F cells. A recombinant, single chain goose FSH was expressed and verified by SDS-PAGE and western blot analysis, and was purified using Protein A agarose affinity and gel filtration chromatography. Biological activity analysis results showed that the recombinant, chimeric goose FSH possesses the function of stimulating estradiol secretion and cell proliferation, in cultured chicken granulosa cells. These results indicated that bioactive, recombinant goose FSH has been successfully prepared in vitro. The recombinant goose FSH will have the potential of being used as a research tool for studying avian reproductive activities, and as a standard for developing avian FSH bioassays.

  18. Single-chain bifunctional vascular endothelial growth factor (VEGF)-follicle-stimulating hormone (FSH)-C-terminal peptide (CTP) is superior to the combination therapy of recombinant VEGF plus FSH-CTP in stimulating angiogenesis during ovarian folliculogenesis.

    PubMed

    Trousdale, Rhonda K; Pollak, Susan V; Klein, Jeffrey; Lobel, Leslie; Funahashi, Yasuhiro; Feirt, Nikki; Lustbader, Joyce W

    2007-03-01

    Infertility technologies often employ exogenous gonadotropin therapy to increase antral follicle production. In an effort to enhance ovarian response, several long-acting FSH therapies have been developed including an FSH-C-terminal peptide (CTP), where the FSH subunits are linked by the CTP moiety from human chorionic gonadotropin, which is responsible for the increased half-life of human chorionic gonadotropin. We found that administration of FSH-CTP for ovarian hyperstimulation in rats blunted ovarian follicle vascular development. In women, reduced ovarian vasculature has been associated with lower pregnancy rates. We were interested in determining whether vascular endothelial growth factor (VEGF) therapy could enhance ovarian angiogenesis in FSH-CTP-treated rats. Coadministration of systemic FSH-CTP plus recombinant VEGF was compared with treatment with a novel, single-chain bifunctional VEGF-FSH-CTP (VFC) analog. For VFC, the FSH portion targets the protein to the ovary and stimulates follicle growth, whereas VEGF enhances local vascular development. Both in vitro and in vivo studies confirm the dual FSH and VEGF action of the VFC protein. Evaluation of ovarian follicle development demonstrates that administration of combination therapy using VEGF and FSH-CTP failed to increase follicle vasculature above levels seen with FSH-CTP monotherapy. However, treatment with VFC significantly increased follicle vascular development while concurrently increasing the number of large antral follicles produced. In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH-CTP protein that is superior to combination therapy for enhancing VEGF activity in the ovary and stimulating follicular angiogenesis in rats.

  19. Follicle-stimulating hormone (FSH) blood test

    MedlinePlus

    ... the test done on certain days of your menstrual cycle. ... In women, FSH helps manage the menstrual cycle and stimulates the ovaries to produce eggs. The test is used to help diagnose or evaluate: Menopause Women who have polycystic ovary ...

  20. Comparisons of mRNA expression for aromatase, FSH receptor, and IGF-I in the granulosa of small ovarian follicles between cattle selected and unselected for twin ovulations

    USDA-ARS?s Scientific Manuscript database

    Long-term selection of cattle for the production of twin ovulations and births has enhanced the development of preantral and antral ovarian follicles and increased the frequency of twin or triplet ovulations to greater than 60%. However, these differences have not been linked to differences in FSH s...

  1. Levels of FSH, LH and testosterone, and sperm DNA fragmentation.

    PubMed

    Wdowiak, Artur; Raczkiewicz, Dorota; Stasiak, Magdalena; Bojar, Iwona

    2014-01-01

    Having an offspring is the most important human biological goal, which is necessary for survival of the human species. Lack of offspring is a phenomenon concerning approximately 15% of married couples in Poland. In a half of the cases, a causative agent is the male factor infertility problem. There is evidence that certain male fertility problems are related with disorders of the process of spermatogenesis. The course of normal spermatogenesis depends on proper pituitary secretion of folliculostimulin (FSH), luteinizing hormone (LH), as well as testicular secretion of testosterone. It is considered that in approximately 20% of patients with idiopathic infertility an elevated level of sperm DNA fragmentation may be the cause of failure in reproduction. The objective of the present study was determination of the relationship between FSH, LH and testosterone levels, and the occurrence of sperm DNA fragmentation. The present study was conducted in the year 2012 in the Non-Public Health Care Unit 'Ovum Reproduction and Andrology' in Lublin, and covered 186 men treated for infertility. For inclusion into the study group we qualified males aged 25-35, who have been treated for infertility for more than 1 year, with no pathological features observed in the female partner. The structure of sperm chromatin was evaluated using the technique of flow cytometry-Sperm Chromatin Structure assay (SCSA). The result of the examination was a sperm DNA Fragmentation Index (DFI), i.e., the percentage of sperm with DNA lesions (DNA fragmentation). A morning blood sample (5 mL volume) was obtained and sent to an authorized laboratory to assess serum levels of testosterone, LH and FSH. An intensified sperm DNA fragmentation co-occurred with both extremely low and extremely high levels of FSH and LH. Sperm DNA fragmentation was negatively correlated with testosterone level.

  2. FSH isoform pattern in classic galactosemia.

    PubMed

    Gubbels, Cynthia S; Thomas, Chris M G; Wodzig, Will K W H; Olthaar, André J; Jaeken, Jaak; Sweep, Fred C G J; Rubio-Gozalbo, M Estela

    2011-04-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

  3. Biosimilar FSH preparations- are they identical twins or just siblings?

    PubMed

    Orvieto, Raoul; Seifer, David B

    2016-06-14

    As patents expire on innovator products, there is increasing interest in developing biosimilar products globally. Biosimilars are not exact copies and are not considered generic versions of the reference product. They may differ in strength, purity and contain different composition of isoforms and/or various glycosylation profiles, with the consequent alterations in clinical efficacy or safety. Recently 2 new recombinant FSH preparations were introduced to clinical practice following randomized controlled, phase 3 clinical trials. Both, Bemfola and Ovaleap® were referred to the FSH innovator product Gonal-f™ (Follitropin alpha), and were found to yield an equivalent number of oocytes (primary end-point), following a long GnRH agonist suppressive protocol in "ideal" patients, i.e., young, normal responders. However, a closer look at these RCTs reveals a non-significant 4 % difference in clinical and ongoing pregnancy rates, in favor of Gonal f over the biosimilar products, accompanied by half the incidence of OHSS (2.9 vs 5.2 %, respectively). These studies were underpowered with reference to pregnancy rates, Thus, we believe that further comparative studies are needed in additional patient populations, e.g.,older,, poor responders, patients with repeated IVF failures and/or polycystic ovary syndrome, before the universal implementation of biosimilar products for clinical use. Biosimilars are actually a regulatory synonym, facilitating a fast track introduction of a FSH preparation to the COH armamentarium. We therefore recommend against interchanging or substituting innovator and biosimilar agents in clinical practice, and believe that the decision whether to use an innovator or a biosimilar product, should be reserved to the discretion of the treating physician. Furthermore, we believe the time has come that the measurement of the biological activity of FSH in humans should require other methods rather than the Steelman-Pohley assay, such as the determination

  4. FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates.

    PubMed

    Grewal, P K; Todd, L C; van der Maarel, S; Frants, R R; Hewitt, J E

    1998-08-17

    The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.

  5. Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation

    PubMed Central

    Chowdhury, Indrajit; Thomas, Kelwyn; Zeleznik, Anthony

    2016-01-01

    Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs. PMID:27044659

  6. Conjugated linoleic acids attenuate FSH- and IGF1-stimulated cell proliferation; IGF1, GATA4, and aromatase expression; and estradiol-17β production in buffalo granulosa cells involving PPARγ, PTEN, and PI3K/Akt.

    PubMed

    Sharma, Isha; Singh, Dheer

    2012-09-01

    Conjugated linoleic acid (CLA) has drawn much interest in last two decades in the area ranging from anticancer activity to obesity. A number of research papers have been published recently with regard to CLA's additional biological functions as reproductive benefits. However, not much is known how this mixture of isomeric compounds mediates its beneficial effects particularly on fertility. In this study, we demonstrated the cross talk between downstream signaling of CLA and important hormone regulators of endocrine system, i.e. FSH and IGF1, on buffalo granulosa cell function (proliferation and steroidogenesis). Experiments were performed in primary serum-free buffalo granulosa cell culture, where cells were incubated with CLA in combination with FSH (25 ng/ml) and IGF1 (50  ng/ml). Results showed that 10 μM CLA inhibits FSH- and IGF1-induced granulosa cell proliferation; aromatase, GATA4, and IGF1 mRNA; and estradiol-17β production. Western blot analysis of total cell lysates revealed that CLA intervenes the IGF1 signaling by decreasing p-Akt. In addition, CLA was found to upregulate peroxisome proliferator-activated receptor-gamma (PPARG) and phosphatase and tensin homolog (PTEN) level in granulosa cells. Further study using PPARG- and PTEN-specific inhibitors supports the potential role of CLA in granulosa cell proliferation and steroidogenesis involving PPARG, PTEN, and PI3K/Akt pathway.

  7. Transcriptional regulation of the FSH receptor: new perspectives

    PubMed Central

    Hermann, Brian P.; Heckert, Leslie L.

    2013-01-01

    The cell-surface receptor for the gonadotropin follicle-stimulating hormone (FSH) is expressed exclusively on Sertoli cells of the testis and granulosa cells of the ovary. FSH signal transduction through its receptor (Fshr) is critical for the timing and maintenance of normal gametogenesis in the mammalian gonad. In the 13 years since the gene encoding Fshr was first cloned, the mechanisms controlling its transcription have been extensively examined, but a clear understanding of what drives its unique cell-specificity remains elusive. Current knowledge of basal Fshr transcription highlights the role of an E-box in the proximal promoter which is bound by the basic helix-loop-helix transcription factors upstream stimulatory factor 1 (Usf1) and Usf2. Recent studies utilizing knockout mice and chromatin immunoprecipitation validated the importance of Usf to Fshr transcription and demonstrated a sexually dimorphic requirement for the Usf proteins to maintain normal Fshr expression. Studies have also shown that the promoter region itself is insufficient for appropriate Fshr expression in transgenic mice, indicating Fshr transcription depends on regulatory elements that lie outside of the promoter. Identification of such elements has been propelled by recent availability of genome sequence data, which facilitated studies using comparative genomics, DNase I hypersensitivity mapping, and transgenic analysis with large fragments of DNA. This review will focus on the current understanding of transcriptional regulatory processes that control expression of rat Fshr, including recent advances from our laboratory. PMID:17084019

  8. Synthesis and characterization of biologically active recombinant elk and horse FSH.

    PubMed

    Fachal, María Victoria; Furlan, Mike; Clark, Rena; Card, Claire E; Chedrese, P Jorge

    2010-02-01

    The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in culture media were characterized by an in vitro bioassay and RIA. When the recombinant products were assessed as activity over mass of FSH measured by RIA, the horse product was 5.6 times more potent than the elk product. The recombinant products injected to immature female Wistar rats stimulated ovarian growth. The results suggest that the products obtained correspond to recombinant versions of the native elk and horse FSH. The availability of these recombinant products may aid in the development of more predictable and efficient techniques of ovarian stimulation in cervids, equids, and other species as well.

  9. Human Lacrimal Gland Gene Expression

    PubMed Central

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  10. NO-mediated regulation of GLUT by T3 and FSH in rat granulosa cells.

    PubMed

    Tian, Ye; Ding, Yu; Liu, Juan; Heng, Dai; Xu, Kaili; Liu, Wenbo; Zhang, Cheng

    2017-03-17

    Thyroid hormones (THs) are important for normal reproductive function. Although 3,5,3'-triiodothyronine (T3) enhances follicle-stimulating hormone (FSH)-induced preantral follicle growth and granulosa cells development in vitro, little is known about the molecular mechanisms regulating ovarian development via glucose. In this study, we investigated whether and how T3 combines with FSH to regulate glucose transporter protein (GLUT) expression and glucose uptake in granulosa cells. Here, we present evidence that T3 and FSH co-treatment significantly increased GLUT-1/GLUT-4 expression, and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of NOS3 expression, total NOS and NOS3 activity and NO content in granulosa cells. Furthermore, we found that activation of the mTOR and PI3K/Akt pathway is required for the regulation of GLUT expression, translocation, and glucose uptake by hormones. We also found that L-arginine (L-arg) up-regulated GLUT-1/GLUT-4 expression and translocation, which were related to increased glucose uptake, however, these responses were significantly blocked by L-NAME. In addition, inhibiting NO production attenuated T3 and FSH-induced GLUT expression, translocation, and glucose uptake in granulosa cells. Our data demonstrate that T3 and FSH co-treatment potentiates cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the mTOR/PI3K/Akt pathway. Meanwhile, NOS3/NO are also involved in this regulatory system. These findings suggest that GLUT is a novel mediator of T3 and FSH-induced follicular development.

  11. Pituitary sex hormones enhance the pro-metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase-1

    PubMed Central

    Abdelbaset-Ismail, Ahmed; Pedziwiatr, Daniel; Schneider, Gabriela; Niklinski, Jacek; Charkiewicz, Radoslaw; Moniuszko, Marcin; Kucia, Magda; Ratajczak, Mariusz Z.

    2017-01-01

    We report that human lung cancer cell lines express functional receptors for pituitary sex hormones (SexHs) and respond to stimulation by follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL). Expression of these receptors has also been confirmed in patient lung cancer samples at the mRNA level. Stimulation of human lung cancer cell lines with FSH, LH, or PRL stimulated migration and chemotaxis, and some cell lines responded by enhanced proliferation. Moreover, priming of human lung cancer cells by exposing them to pituitary SexHs resulted in enhanced seeding efficiency of injected human lung cancer cells into bone marrow, liver, and lungs in an immunodeficient mouse model. The chemotaxis of lung cancer cell lines corresponded with the activity of heme oxygenase-1 (HO-1), as stimulation of these cells by FSH, LH, and PRL downregulated its expression in a p38 MAPK-dependent manner. Moreover, while downregulation of HO-1 by the small-molecule inhibitor tin protoporphyrin (SnPP) promoted migration, upregulation of HO-1 by the small-molecule activator cobalt protoporphyrin (CoPP) showed the opposite effect. Based on this finding, we propose that pituitary SexHs play a significant role in the pathogenesis of lung cancer, particularly when the blood level of FSH increases due to gonadal dysfunction with advanced age. Finally, we propose that upregulation of HO-1 expression by a small-molecule activator may be effective in controlling SexH-induced cell migration in lung cancer. PMID:27922667

  12. Fsh and Lh direct conserved and specific pathways during flatfish semicystic spermatogenesis.

    PubMed

    Chauvigné, François; Zapater, Cinta; Crespo, Diego; Planas, Josep V; Cerdà, Joan

    2014-10-01

    The current view of the control of spermatogenesis by Fsh and Lh in non-mammalian vertebrates is largely based on studies carried out in teleosts with cystic and cyclic spermatogenesis. Much less is known concerning the specific actions of gonadotropins during semicystic germ cell development, a type of spermatogenesis in which germ cells are released into the tubular lumen where they transform into spermatozoa. In this study, using homologous gonadotropins and a candidate gene approach, for which the genes' testicular cell-type-specific expression was established, we investigated the regulatory effects of Fsh and Lh on gene expression during spermatogenesis in Senegalese sole (Solea senegalensis), a flatfish with asynchronous and semicystic germ cell development. During early spermatogenesis, Fsh and Lh upregulated steroidogenesis-related genes and nuclear steroid receptors, expressed in both somatic and germ cells, through steroid-dependent pathways, although Lh preferentially stimulated the expression of downstream genes involved in androgen and progestin syntheses. In addition, Lh specifically promoted the expression of spermatid-specific genes encoding spermatozoan flagellar proteins through direct interaction with the Lh receptor in these cells. Interestingly, at this spermatogenic stage, Fsh primarily regulated genes encoding Sertoli cell growth factors with potentially antagonistic effects on germ cell proliferation and differentiation through steroid mediation. During late spermatogenesis, fewer genes were regulated by Fsh or Lh, which was associated with a translational and posttranslational downregulation of the Fsh receptor in different testicular compartments. These results reveal that conserved and specialized gonadotropic pathways regulate semicystic spermatogenesis in flatfish, which may spatially adjust cell germ development to maintain a continuous reservoir of spermatids in the testis.

  13. Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

    SciTech Connect

    Schreiber, J.R.; Nakamura, K.; Schmit, V.; Weinstein, D.B.

    1984-01-01

    Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, the authors examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with /sup 125/I-lipoproteins (human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)). The media were then analyzed for lipoprotein protein coat degradation products (mainly /sup 125/I-monoiodotyrosine) and progestin (mainly 20 alpha-dihydroprogesterone (20 alpha-DHP)). In the absence of FSH and androgen, 2 X 10(5) granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20 alpha-DHP. The addition of 10(-7) M androstenedione (A), testosterone (T), or 5 alpha-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20 alpha-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20 alpha-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20 alpha-DHP production.

  14. Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca).

    PubMed

    Liao, Ming-Juan; Zhu, Mu-Yuan; Zhang, Zhi-He; Zhang, An-Ju; Li, Guang-Han; Sheng, Fu-Jun

    2003-05-15

    The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.

  15. Pubertal Onset in Girls is Strongly Influenced by Genetic Variation Affecting FSH Action

    PubMed Central

    Hagen, Casper P.; Sørensen, Kaspar; Aksglaede, Lise; Mouritsen, Annette; Mieritz, Mikkel G.; Tinggaard, Jeanette; Wohlfart-Veje, Christine; Petersen, Jørgen Holm; Main, Katharina M.; Meyts, Ewa Rajpert-De; Almstrup, Kristian; Juul, Anders

    2014-01-01

    Age at pubertal onset varies substantially in healthy girls. Although genetic factors are responsible for more than half of the phenotypic variation, only a small part has been attributed to specific genetic polymorphisms identified so far. Follicle-stimulating hormone (FSH) stimulates ovarian follicle maturation and estradiol synthesis which is responsible for breast development. We assessed the effect of three polymorphisms influencing FSH action on age at breast deveopment in a population-based cohort of 964 healthy girls. Girls homozygous for FSHR -29AA (reduced FSH receptor expression) entered puberty 7.4 (2.5–12.4) months later than carriers of the common variants FSHR -29GG+GA, p = 0.003. To our knowledge, this is the strongest genetic effect on age at pubertal onset in girls published to date. PMID:25231187

  16. Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

    PubMed

    Nóbrega, Rafael Henrique; Morais, Roberto Daltro Vidal de Souza; Crespo, Diego; de Waal, Paul P; de França, Luiz Renato; Schulz, Rüdiger W; Bogerd, Jan

    2015-10-01

    Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

  17. [Low-dose desmopressin (DDAVP) and blood levels of FSH, LH and testosterone in men].

    PubMed

    García-Pascual, I J; Rozán Flores, M A

    1996-03-01

    The effect of desmopressin (DDAVP) administration (2.5 micrograms/12 hours) on serum concentrations of FSH, LH and testosterone was studied in six men. No significant changes were observed in serum concentrations of FSH and LH after 9 days with DDAVP therapy. Nevertheless, serum concentrations of testosterone after 12 hours of DDAVP administration were significantly higher than basal concentrations. Three hours after the administration of DDAVP, serum testosterone concentrations decreased significantly. The conclusion reached was that low doses of desmopressin do not change serum concentrations of FSH and LH, but serum concentration of testosterone is decreased within three hours after the administration, although an increase is observed 12 hours later possibly due to a "rebound effect". Desmopressin would therefore directly act upon human testicle.

  18. Accelerated growth of bovine preantral follicles in vitro after stimulation with both FSH and BMP-15 is accompanied by ultrastructural changes and increased atresia.

    PubMed

    Passos, M J; Vasconcelos, G L; Silva, A W B; Brito, I R; Saraiva, M V A; Magalhães, D M; Costa, J J N; Donato, M A M; Ribeiro, R P; Cunha, E V; Peixoto, C A; Campello, C C; Figueiredo, J R; van den Hurk, R; Silva, J R V

    2013-06-01

    The objective of the present study was to determine the effects of bone morphogenetic protein (BMP)-15 and FSH on the growth, viability, and expression of mRNA for FSH (FSH-R) and BMP-15 (BMPR-IB and BMPR-II) receptors in cultured bovine secondary follicles. Secondary follicles were microdissected and cultured for 12 days in minimum essential medium-α alone or supplemented with BMP-15, sequential FSH, both BMP-15 and FSH, or BMP-15 from days 0 to 6, and FSH from days 7 to 12. Thereafter, the effect of these treatments on the follicular volume, viability, and antrum formation and the levels of mRNA for BMPR-IB, BMPR-II, and FSH-R were assessed. Compared with day 0, the follicles cultured with FSH or BMP-15, or both, had a significant and progressive increase in volume (P < 0.05). However, the follicles cultured for 12 days with both BMP-15 and FSH had the greatest volume and a greater rate of antrum formation than those in control medium, but results similar to those cultured with FSH (days 0 to 12) or BMP-15 (days 0 to 6) and FSH (days 7 to 12). Together with their accelerating effect on in vitro follicle growth, the combination of FSH and BMP-15 induced ultrastructural changes in the cultured follicles and increased atresia. However, adding either BMP-15 or FSH to the culture medium, not only promoted follicular growth and follicular antrum formation, but also maintained follicular viability during culture. Except for follicles cultured in minimal essential medium-α, the levels of mRNA for BMPR-IB were reduced, and the levels of mRNA for FSH-R were significantly greater in follicles cultured in medium supplemented with BMP-15. In conclusion, all in vitro follicle treatments supported growth of bovine preantral follicles; however, adding both BMP-15 and FSH to the culture medium (minimal essential medium-α) for 12 days provided the greatest stimulation. Furthermore, the viability and ultrastructural integrity of cultured follicles were only maintained when

  19. Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism.

    PubMed

    Ruvolo, Giovanni; Roccheri, Maria Carmela; Brucculeri, Anna Maria; Longobardi, Salvatore; Cittadini, Ettore; Bosco, Liana

    2013-04-01

    An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p<0.05), but we found a significant reduction in patients with high basal DFI values (>15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %. Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .

  20. The TATA Binding Protein Associated Factor 4b (TAF4b) Mediates FSH Stimulation of the IGFBP-3 Promoter in Cultured Porcine Ovarian Granulosa Cells

    PubMed Central

    Ongeri, Elimelda Moige; Verderame, Michael F.; Hammond, James M.

    2007-01-01

    We have established the gene for IGF binding protein 3 (IGFBP-3) as a target for FSH action. FSH effects on this gene require the PKA pathway as well as the PI-3 kinase and MAPK pathways. At the IGFBP-3 promoter, FSH effects depend on a site for TATA box binding protein (TBP) and formation of a high molecular weight transcription complex. To further elucidate FSH effects on the downstream events involving the TBP site, we cloned a pig TAF4b cDNA into a P-Flag expression vector. By co-transfecting granulosa cells with the IGFBP-3 promoter, we found that TAF4b mimics and enhances FSH induction of IGFBP-3 reporter activity. Using RT-PCR we showed that FSH stimulates expression of TAF4b. This would suggest that the role of TAF4b in follicular development is regulated by FSH. TAF4b may thus be the TFIID component that binds to the TBP site on the IGFBP-3 promoter and is essential for FSH induction of IGFBP-3. PMID:17888567

  1. High levels of testosterone inhibit ovarian follicle development by repressing the FSH signaling pathway.

    PubMed

    Liu, Tao; Cui, Yu-qian; Zhao, Han; Liu, Hong-bin; Zhao, Shi-dou; Gao, Yuan; Mu, Xiao-li; Gao, Fei; Chen, Zi-jiang

    2015-10-01

    The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.

  2. Determination of biological activity of gonadotropins hCG and FSH by Förster resonance energy transfer based biosensors.

    PubMed

    Mazina, Olga; Allikalt, Anni; Tapanainen, Juha S; Salumets, Andres; Rinken, Ago

    2017-02-09

    Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein (T)Epac(VV) in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.

  3. Determination of biological activity of gonadotropins hCG and FSH by Förster resonance energy transfer based biosensors

    PubMed Central

    Mazina, Olga; Allikalt, Anni; Tapanainen, Juha S.; Salumets, Andres; Rinken, Ago

    2017-01-01

    Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein TEpacVV in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research. PMID:28181555

  4. The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH.

    PubMed

    Sacchi, Sandro; D'Ippolito, Giovanni; Sena, Paola; Marsella, Tiziana; Tagliasacchi, Daniela; Maggi, Elena; Argento, Cindy; Tirelli, Alessandra; Giulini, Simone; La Marca, Antonio

    2016-01-01

    Anti Müllerian Hormone (AMH) has a negative and inhibitory role in many functions of human granulosa-lutein cells (hGCs) including notoriously the reduction of the aromatase CYP19A1 expression induced by follicle-stimulating hormone (FSH). No data have been provided on the possible role of AMH in modulating the response to luteinizing hormone (LH) (alone or combined with FSH) as well as its effect on other enzymes involved in steroidogenesis including aromatase P450scc. The aim of this study was to investigate the role of AMH as regulator of the basal and stimulated steroids production by hGCs. Primary culture of hGCs were incubated with hormones AMH, LH, and FSH, alone or in combination. The CYP19A1 and P450scc messenger RNA (mRNA) expression, normalized by housekeeping ribosomal protein S7 (RpS7) gene, was evaluated by reverse transcriptase quantitative PCR (RT-qPCR). Each reaction was repeated in triplicate. Negative controls using corresponding amount of vehicle control for each hormone treatment were performed. AMH did not modulate the basal mRNA expression of both aromatase genes at any of the concentrations tested. Meanwhile, the strong mRNA induction of CYP19A1 and P450scc generated by a 24-h gonadotropin treatment (alone and combined) was suppressed by 20 ng/ml AMH added to culture medium. These findings contribute in clarifying the relationship between hormones regulating the early phase of steroidogenesis confirming that AMH is playing a suppressive role on CYP19A1 expression stimulated by gonadotropin in hGCs. Furthermore, a similar inhibitory effect for AMH was observed on P450scc gene expression when activated by gonadotropin treatment.

  5. A double-bromodomain protein, FSH-S, activates the homeotic gene ultrabithorax through a critical promoter-proximal region.

    PubMed

    Chang, Yuh-Long; King, Balas; Lin, Shu-Chun; Kennison, James A; Huang, Der-Hwa

    2007-08-01

    More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic (fs(1)h) gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. We show that one of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. First, overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation. Second, FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner. Third, purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, we show that FSH-S is involved in phosphorylation of itself and other regulatory factors. We suggest that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers.

  6. FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function.

    PubMed

    Banerjee, Antara A; Dupakuntla, Madhavi; Pathak, Bhakti R; Mahale, Smita D

    2015-06-01

    The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH-FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L(501)F mainly and I(505)V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L(501)F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.

  7. Recombinant human follicle-stimulating hormone and transforming growth factor-alpha enhance in vitro maturation of porcine oocytes.

    PubMed

    Mito, Tomomi; Yoshioka, Koji; Noguchi, Michiko; Yamashita, Shoko; Hoshi, Hiroyoshi

    2013-07-01

    The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1 IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1 IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20 hr culture and HAPLN1 mRNA after 44 hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways.

  8. Differential actions of FSH and LH during folliculogenesis.

    PubMed

    Palermo, Roberto

    2007-09-01

    In the gonadotrophin-dependent stage of follicular development, FSH- and LH-signalling pathways play an obligatory role in follicle differentiation, selection and survival. Under the effect of LH the theca-interstitial cell layer acts as an androgen producer. Thus, androgen diffusing into the mural granulosa cell layer represents the substrate for FSH-induced aromatase for follicular oestradiol synthesis. This is the landmark 'two cell-two gonadotrophin' concept in the physiology of ovarian function in mammals. The increase in plasma FSH during luteo-follicular transition is the basis for follicle selection. The rise of FSH to the threshold concentration represents a critical condition for the growth of the most sensitive follicle in a given time frame of the last 14 days of the dominant follicle odyssey. The gonadotrophin-induced follicular oestradiol secretion inhibits pituitary secretion of FSH, which in turn causes the concentration of FSH in the developing cohort follicles to drop below threshold concentrations and the arrest of the development of the less FSH-sensitive follicle (FSH threshold and window concept). In the gonadotrophin-dependent phase of follicular development, LH also seems to acts within a critical window of the hormone concentration framed between the minimal threshold and a ceiling for the normal functions of the follicle unit.

  9. Phosphoinositide 3-kinase p110δ mediates estrogen- and FSH-stimulated ovarian follicle growth.

    PubMed

    Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing; Fan, Heng-Yu; Sha, Jiahao

    2013-09-01

    In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

  10. Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

    SciTech Connect

    Ford, K.A.; LaBarbera, A.R.

    1988-11-01

    The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase in receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.

  11. Sphingosine-1-phosphate, regulated by FSH and VEGF, stimulates granulosa cell proliferation.

    PubMed

    Hernández-Coronado, C G; Guzmán, A; Rodríguez, A; Mondragón, J A; Romano, M C; Gutiérrez, C G; Rosales-Torres, A M

    2016-09-15

    Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro. Seventy five thousand bovine granulosa cells from healthy medium-sized (4-7mm) follicles were cultured in 96-well plates in McCoy's 5a medium containing 10ng/mL of insulin and 1ng/mL of LR-IGF-I at 37°C in a 5% CO2/air atmosphere at 37°C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96h, with the maximum production observed with 1ng/mL of FSH. Likewise, 0.01ng/mL of VEGF stimulated S1P production at 48, but not 96h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48h of culture. FSH increased the expression of phosphorylated SK1 (P<0.05) and the addition of a SK1 inhibitor reduced the constitutive and FSH-stimulated S1P synthesis (P<0.05). Sphingosine-1-phosphate had a biphasic effect on granulosa cell number after culture. At low concentration S1P (0.1μM) increased granulosa cell number after 48h of culture (P<0.05) and the proportion of cells in the G2 and M phase of the cell cycle (P<0.05), whereas higher concentrations decreased cell number (10μM; P<0.05) by an increase (P<0.05) in the

  12. Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation.

    PubMed

    Dupakuntla, Madhavi; Pathak, Bhakti; Roy, Binita Sur; Mahale, Smita D

    2012-10-15

    The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling.

  13. Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition.

    PubMed

    Orisaka, Makoto; Hattori, Katsushige; Fukuda, Shin; Mizutani, Tetsuya; Miyamoto, Kaoru; Sato, Takashi; Tsang, Benjamin K; Kotsuji, Fumikazu; Yoshida, Yoshio

    2013-08-01

    Several clinical studies have shown a correlation of hypersecretion of LH and polycystic ovary syndrome (PCOS), infertility, and miscarriage in women, suggesting that chronically elevated LH impairs fertility. Growth arrest of small antral follicles in PCOS is also assumed to be associated with an abnormal endocrine environment involving increased LH stimulation, a hyperandrogenic milieu, and subsequent dysregulated FSH action in the ovarian follicles. In this study, we examined whether and how LH modulates follicular development and steroid production during preantral-early antral follicle transition by using a rat preantral follicle culture system. LH augments testosterone and estradiol production in preantral follicles via up-regulating mRNA abundance of CYP17A1 and CYP19A1. LH promotes rat preantral follicle growth, and the follicular size reaches that of early antral follicles in vitro, a response attenuated by the specific androgen receptor antagonist and a targeted disruption of androgen receptor gene. Sustained follicle stimulation by LH, but not by androgen, decreases FSH receptor mRNA levels and FSH receptor signaling and inhibits FSH-induced follicular growth. The data suggest that LH promotes preantral-early antral transition via the increased synthesis and growth-promoting action of androgen. However, chronic LH stimulation impairs FSH-dependent antral follicle growth by suppressing granulosa cell FSHR expression via the modulation of intraovarian regulators, including LH-induced thecal factors.

  14. FSH-stimulated follicular secretions enhance oocyte maturation in pigs.

    PubMed

    Ding, J; Foxcroft, G R

    1994-01-01

    Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.

  15. Human rhabdomyosarcoma cells express functional pituitary and gonadal sex hormone receptors: Therapeutic implications

    PubMed Central

    PONIEWIERSKA-BARAN, AGATA; SCHNEIDER, GABRIELA; SUN, WENYUE; ABDELBASET-ISMAIL, AHMED; BARR, FREDERIC G.; RATAJCZAK, MARIUSZ Z.

    2016-01-01

    Evidence has accumulated that sex hormones play an important role in several types of cancer. Because they are also involved in skeletal muscle development and regeneration, we were therefore interested in their potential involvement in the pathogenesis of human rhabdomyosarcoma (RMS), a skeletal muscle tumor. In the present study, we employed eight RMS cell lines (three fusion positive and five fusion negative RMS cell lines) and mRNA samples obtained from RMS patients. The expression of sex hormone receptors was evaluated by RT-PCR and their functionality by chemotaxis, adhesion and direct cell proliferation assays. We report here for the first time that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors are expressed in established human RMS cell lines as well as in primary tumor samples isolated from RMS patients. We also report that human RMS cell lines responded both to pituitary and gonadal sex hormone stimulation by enhanced proliferation, chemotaxis, cell adhesion and phosphorylation of MAPKp42/44 and AKT. In summary, our results indicate that sex hormones are involved in the pathogenesis and progression of RMS, and therefore, their therapeutic application should be avoided in patients that have been diagnosed with RMS. PMID:26983595

  16. Neuropharmacology of Human Appetite Expression

    ERIC Educational Resources Information Center

    Halford, Jason C. G.; Harrold, Joanne A.

    2008-01-01

    The regulation of appetite relies on the integration of numerous episodic (meal) and tonic (energy storage) generated signals in energy regulatory centres within the central nervous system (CNS). These centers provide the pharmacological potential to modify human appetite (hunger and satiety) to increase or decrease caloric intake, or to normalize…

  17. Neuropharmacology of Human Appetite Expression

    ERIC Educational Resources Information Center

    Halford, Jason C. G.; Harrold, Joanne A.

    2008-01-01

    The regulation of appetite relies on the integration of numerous episodic (meal) and tonic (energy storage) generated signals in energy regulatory centres within the central nervous system (CNS). These centers provide the pharmacological potential to modify human appetite (hunger and satiety) to increase or decrease caloric intake, or to normalize…

  18. Construction and expression of recombined human AFP eukaryotic expression vector

    PubMed Central

    Zhang, Li-Wang; Ren, Jun; Zhang, Liang; Zhang, Hong-Mei; Jin, Bin; Pan, Bo-Rong; Si, Xiao-Ming; Zhang, Yan-Jun; Wang, Zhong-Hua; Pan, Yang-Lin; Festein, Stephen M

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/mL AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P < 0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma. PMID:12854142

  19. TPRV-1 expression in human preeclamptic placenta.

    PubMed

    Martínez, Nora; Abán, Cyntia E; Leguizamón, Gustavo F; Damiano, Alicia E; Farina, Mariana G

    2016-04-01

    Preeclampsia is a multisystem disorder unique to human pregnancy, characterized by abnormal placentation. Although its causes remain unclear, it is known that the expression of several transporters is altered. Transient receptor potential vanilloid 1 (TRPV-1) is a nonselective cation channel, present in human placenta. Here, we evaluated the expression of TRPV-1 in preeclamptic placentas. We observed a deregulation in TRPV-1 expression in these placentas which may explain the impaired Ca(2+) homeostasis found in preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Expression of polarity genes in human cancer.

    PubMed

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  1. Expression of Polarity Genes in Human Cancer

    PubMed Central

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical–basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function. PMID:25991909

  2. Clusterin expression and human testicular seminoma.

    PubMed

    Tang, Min; Li, Jie; Liu, Bianjiang; Song, Ninghong; Wang, Zengjun; Yin, Changjun

    2013-10-01

    Clusterin expression has a positive correlation with the occurrence and progression of various types of tumors from different genetic backgrounds. Clusterin overexpression may protect tumor cells from apoptosis and damage caused by autoimmunity or anti-tumor therapy. Using immunohistochemisty, one previous study showed that clusterin protein expression is downregulated in human testicular seminoma, which is highly sensitive to radiotherapy and chemotherapy. We thus postulate that clusterin expression in human testicular seminoma differs from clusterin expression in other tumors. It may be the cause of the treatable characteristics of testicular seminoma. In the present preliminary study, we detected the abundance of clusterin mRNA in human testicular seminoma and normal testis. The results showed decreased clusterin expression in seminoma at the gene transcription level. Our primary data and summarized previous literature suggest that the downregulation of clusterin expression may be the cause of the high sensitivity of testicular seminoma to radiotherapy and chemotherapy. It may be that the scarcity of clusterin leaves tumor cells with insufficient protection from treatment. This is the first study to focus on the relationship between clusterin expression and human testicular cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Expression of prostacyclin receptor in human megakaryocytes.

    PubMed

    Sasaki, Y; Takahashi, T; Tanaka, I; Nakamura, K; Okuno, Y; Nakagawa, O; Narumiya, S; Nakao, K

    1997-08-01

    Prostacyclin (prostaglandin I2, PGI2) is a potent vasodilator and inhibitor of platelet aggregation. Although it is well known that the specific receptor for prostacyclin (PGI2-R) is abundantly expressed on platelets, PGI2-R expression in megakaryocytes is poorly understood. In this study, we examined its expression in leukemic or normal megakaryocytes. PGI2-R mRNA was expressed in human leukemic cell lines of megakaryocytic nature as evaluated by Northern blot analysis. Phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), and tumor necrosis factor-alpha (TNF-alpha) enhanced PGI2-R mRNA expression. The enhancement of PGI2-R expression by PMA and TPO was associated with the upregulation of platelet factor 4 or glycoprotein IIb mRNA expression. Iloprost, an agonist of prostacyclin, induced significant cyclic (c)AMP synthesis in these leukemic cells indicating that interaction of PGI2-R and its ligand can induce postreceptor signal transduction. Furthermore, iloprost-induced cAMP synthesis was enhanced by the pretreatment with PMA or the cytokines that promoted PGI2-R expression. PMA and TPO also increased the specific binding of [3H]iloprost to these cells. Pooled normal megakaryocytic colonies from TPO-containing semisolid culture of purified human CD34+ cells expressed PGI2-R, which were increased as the megakaryocytes matured with the peak expression before proplatelet formation, as evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These results indicate that PGI2-R is expressed in human megakaryocytes and is upregulated by cytokines involved in thrombopoiesis or inflammation. Also, it was indicated that megakaryocytic maturation accompanies enhancement of PGI2-R expression.

  4. Heterogeneity of rat FSH by chromatofocusing: studies on serum FSH, hormone released in vitro and metabolic clearance rates of its various forms.

    PubMed

    Blum, W F; Gupta, D

    1985-04-01

    Rat pituitary FSH was fractionated by chromatofocusing between pH 6 and 3. Ten components were resolved having apparent isoelectric points between 3.1 and 5.1. A comparative study of pituitary FSH and FSH secreted in vitro by quartered pituitary glands in the presence and in the absence of gonadotrophin-releasing hormone (GnRH) revealed similar patterns of charged species of intracellular and released FSH. Although GnRH increased FSH secretion about fourfold, no influence on the pattern of charged species was observed. Utilizing exclusion chromatography and chromatofocusing, pituitary FSH was compared to serum FSH which had been extracted by immuno-affinity chromatography. The results demonstrate for serum FSH a larger molecular size and a relative shift to more acidic components. Metabolic clearance rates of eight FSH components separated by chromatofocusing were measured in adult male rats. Half-lives varied between 13 min and several hours. A correlation existed between decrease of isoelectric points and decrease of metabolic clearance rates. These findings suggest that all hypophysial FSH components are secreted into the circulation at similar rates and the more acidic FSH components which appear to contain increased sialic acid, have a longer circulatory half-life and are more abundant in serum. It is concluded that sialylation may be involved in modulating serum FSH levels.

  5. Single-cell gene expression analysis reveals diversity among human spermatogonia.

    PubMed

    Neuhaus, N; Yoon, J; Terwort, N; Kliesch, S; Seggewiss, J; Huge, A; Voss, R; Schlatt, S; Grindberg, R V; Schöler, H R

    2017-02-10

    Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. The heterogeneity of human

  6. Costs and outcomes associated with IVF using recombinant FSH.

    PubMed

    Ledger, W; Wiebinga, C; Anderson, P; Irwin, D; Holman, A; Lloyd, A

    2009-09-01

    Cost and outcome estimates based on clinical trial data may not reflect usual clinical practice, yet they are often used to inform service provision and budget decisions. To expand understanding of assisted reproduction treatment in clinical practice, an economic evaluation of IVF/intracytoplasmic sperm injection (ICSI) data from a single assisted conception unit (ACU) in England was performed. A total of 1418 IVF/ICSI cycles undertaken there between October 2001 and January 2006 in 1001 women were analysed. The overall live birth rate was 22% (95% CI: 19.7-24.2), with the 30- to 34-year age group achieving the highest rate (28%). The average recombinant FSH (rFSH) dose/cycle prescribed was 1855 IU. Average cost of rFSH/cycle was 646 pound(SD: 219 pound), and average total cost/cycle was 2932 pound (SD: 422 pound). Economic data based on clinical trials informing current UK guidance assumes higher doses of rFSH dose/cycle (1750-2625 IU), higher average cost of drugs/cycle (1179 pound), and higher average total cost/cycle (3266 pound). While the outcomes in this study matched UK averages, total cost/cycle was lower than those cited in UK guidelines. Utilizing the protocols and (lower) rFSH dosages reported in this study may enable other ACU to provide a greater number of IVF/ICSI cycles to patients within given budgets.

  7. IL-21 Receptor Expression in Human Tendinopathy

    PubMed Central

    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  8. FSH up-regulates angiogenic factors in luteal cells of buffaloes.

    PubMed

    Fátima, L A; Evangelista, M C; Silva, R S; Cardoso, A P M; Baruselli, P S; Papa, P C

    2013-11-01

    Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4-6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was

  9. Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

    PubMed

    Guzmán, J M; Rubio, M; Ortiz-Delgado, J B; Klenke, U; Kight, K; Cross, I; Sánchez-Ramos, I; Riaza, A; Rebordinos, L; Sarasquete, C; Zohar, Y; Mañanós, E L

    2009-07-01

    The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

  10. TIE-2 expressing monocytes in human cancers

    PubMed Central

    Pabois, Angélique; Xenarios, Ioannis; Delaloye, Jean-François; Doucey, Marie-Agnès

    2017-01-01

    ABSTRACT Tumor-associated macrophages (TAM) are well known as a key player in the tumor microenvironment, which support cancer progression. More recently, a lineage of monocytes characterized by the expression of the TIE-2/Tek angiopoietin receptor identified a subset of circulating and tumor-associated monocytes endowed with proangiogenic activity. TIE-2 expressing monocytes (TEM) were found both in humans and mice. Here, we review the phenotypes and functions of TEM reported so far in human cancer and their potential use as markers of cancer progression and metastasis. Finally, we discuss the therapeutic approaches currently used or proposed to target TEM. PMID:28507810

  11. Whole brain-pituitary in vitro preparation of the transgenic medaka (Oryzias latipes) as a tool for analyzing the differential regulatory mechanisms of LH and FSH release.

    PubMed

    Karigo, Tomomi; Aikawa, Masato; Kondo, Chika; Abe, Hideki; Kanda, Shinji; Oka, Yoshitaka

    2014-02-01

    Two types of gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are important pituitary hormones for sexual maturation and reproduction, and both of them are centrally regulated by gonadotropin-releasing hormone (GnRH) from the hypothalamus. In mammals, these two gonadotropins are secreted from a single type of gonadotrope. The mechanisms of differential regulation by GnRH of the release of two types of gonadotropins with different secretory profiles are still unknown. In teleosts, however, LH and FSH are secreted from separate cellular populations, unlike in mammals. This feature makes them useful for studying the regulatory mechanisms of LH and FSH secretions independently. Here, we generated transgenic medaka lines that express Ca(2+) indicator protein, inverse-pericam, specifically in the LH or FSH cells. We performed cell-type-specific Ca(2+) imaging of LH and FSH cells, respectively, using the whole brain-pituitary preparations of these transgenic fish in which all neural circuits and GnRH neuronal projection to the pituitary are kept intact. LH and FSH cells showed different Ca(2+) responses to GnRH. The results suggest differential regulation mechanisms for LH and FSH release by GnRH. Moreover, we also succeeded in detecting the effect on LH cells of endogenous GnRH peptide, which was released by electrical stimulation of the axons of GnRH1 neurons. Thus, our newly developed experimental model system using the whole brain-pituitary in vitro preparation of the transgenic medaka is a powerful tool for analyzing the differential regulatory mechanisms of the release of LH and FSH by multisynaptic neural inputs to the pituitary.

  12. Thyrotropin-releasing hormone provokes abnormal follicle-stimulating hormone (FSH) and luteinizing hormone responses in men who have pituitary adenomas and FSH hypersecretion.

    PubMed

    Snyder, P J; Muzyka, R; Johnson, J; Utiger, R D

    1980-10-01

    Serum FSH ad LH concentrations after the administration of TRH were measured in 10 men who had pituitary adenomas associated with FSH hypersecretion. Similar measurements were made in 12 men who had pituitary adenomas but no FSH hypersecretion, in 10 age-matched, normal men, and in 5 men who had primary hypergonadism. The mean serum LH concentration in the men who had pituitary adenomas and FSH hypersecretion increased 136% after TRH administration, significantly greataer (P < 0.005) than the 48% increase in the normal men or the 51% increase in the men who had pituitary adenomas without FSH hypersecretion. Serum LH did not increase at all in the men who had primary hypoganadism. The serum FSH concentration did not increase in any of the normal men, in the men who had pituitary adenomas without FSH hypersecretion, or in the men who had primary hypogonadism, but did increase in 5 of the 10 men who had FSH hypersecretion; the mean increase in these 5 men was 38%. The exaggerated LH responses and the nonspecific FSH responses to TRH of the men who had pituitary adenomas associated with FSH hypersecretion suggest that control of both FSH and LH secretion by these adenomas is abnormal and, therefore, that these adenomas are likely gonadotroph cell adenomas.

  13. Human eosinophils express functional CCR7.

    PubMed

    Akuthota, Praveen; Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F

    2013-06-01

    Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5-primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM-loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5-primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5-primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7.

  14. Delayed puberty and primary amenorrhea associated with a novel mutation of the human follicle-stimulating hormone receptor: clinical, histological, and molecular studies.

    PubMed

    Meduri, G; Touraine, P; Beau, I; Lahuna, O; Desroches, A; Vacher-Lavenu, M C; Kuttenn, F; Misrahi, M

    2003-08-01

    Inactivating mutations of the FSH receptor have been described in rare cases of premature ovarian failure. Only one mutation was associated with a complete phenotype, including delayed puberty, primary amenorrhea, and small ovaries. We describe here a new patient presenting a similar complete phenotype of premature ovarian failure, with high plasma FSH levels associated with very low estrogen and inhibin B levels. No biological response to high doses of recombinant FSH was detected. A novel homozygous Pro(519)Thr mutation was found in this patient. This mutation is located in the second extracellular loop of the FSH receptor, within a motif highly conserved in gonadotropin and TSH receptors. The mutation totally impairs adenylate cyclase stimulation in vitro. FSH binding experiments and confocal microscopy showed that this mutation alters the cell surface targeting of the mutated receptor, which remains trapped intracellularly. Histological studies of the ovaries of the patient showed an increase in the density of small follicles compared with age-matched normal women. A complete block in follicular maturation after the primary stage was also observed. Immunocytochemical studies allowed detection of the expression of c-Kit and proliferation cellular nuclear antigen, whereas no apoptosis was shown by the 3'-end-labeling method. This observation supports the concept that in humans FSH seems mandatory for the initiation of follicular growth only after the primary stage. In our patient complete FSH resistance yields infertility, which is remarkably associated with the persistence of a high number of small follicles.

  15. [Facial expressions and human emotions. Bibliographic survey].

    PubMed

    da Silva, J A; da Silva, M J

    1995-01-01

    The present work intends to divulge some Nursing and Psychology' publications about human facies and emotions. Intends do help the health professional thinking about the facial expressions of emotion in the interpersonal relationships, showing the researches more cited at specific bibliography, first regarding to children and to adults.

  16. Expression cloning of human B cell immunoglobulins.

    PubMed

    Wardemann, Hedda; Kofer, Juliane

    2013-01-01

    The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

  17. CD161-expressing human T cells.

    PubMed

    Fergusson, Joannah R; Fleming, Vicki M; Klenerman, Paul

    2011-01-01

    Expression of the Natural Killer cell receptor CD161 has recently been identified on a subset of T cells, including both CD4+ T helper and CD8+ T cells. Expression of this molecule within the adult circulation is restricted to those T cells with a memory phenotype. However, the distinct properties of these T cell populations is yet to be fully determined, although expression of CD161 has been related to the secretion of interleukin-17, and therefore to a type 17 phenotype. Recent studies have aimed to determine both the origin of these cells and the significance of CD161 expression as either a marker of specific cell types or as an effector and regulator of lymphocyte function, and hence to characterize the role of these CD161+ cells within a variety of human diseases in which they have been implicated.

  18. Regulation of gene expression in human tendinopathy

    PubMed Central

    2011-01-01

    Background Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. Methods We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. Results Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. Conclusions Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics. PMID:21539748

  19. Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle.

    PubMed

    El-Hayek, Stephany; Demeestere, Isabelle; Clarke, Hugh J

    2014-11-25

    Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb(-/-) mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb(-/-) oocytes to produce essential oocyte-secreted factors or of Fshb(-/-) cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb(+/-) females, these increases fail to occur in Fshb(-/-) females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb(-/-) females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.

  20. Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle

    PubMed Central

    El-Hayek, Stephany; Demeestere, Isabelle; Clarke, Hugh J.

    2014-01-01

    Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb−/− mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb−/− oocytes to produce essential oocyte-secreted factors or of Fshb−/− cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb+/− females, these increases fail to occur in Fshb−/− females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb−/− females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility. PMID:25385589

  1. Concanavalin-A induces granulosa cell death and inhibits FSH-mediated follicular growth and ovarian maturation in female rats.

    PubMed

    Velasquez, Ethel V; Ríos, Mariana; Ortiz, María Elena; Lizama, Carlos; Nuñez, Elizabeth; Abramovich, Dalhia; Orge, Felipe; Oliva, Barbara; Orellana, Renán; Villalon, Manuel; Moreno, Ricardo D; Tesone, Marta; Rokka, Anne; Corthals, Garry; Croxatto, Horacio B; Parborell, Fernanda; Owen, Gareth I

    2013-05-01

    Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

  2. Opsin Expression in Human Epidermal Skin

    PubMed Central

    Haltaufderhyde, Kirk; Ozdeslik, Rana N; Wicks, Nadine L; Najera, Julia A; Oancea, Elena

    2015-01-01

    Human skin is constantly exposed to solar light containing visible and ultraviolet radiation (UVR), a powerful skin carcinogen. UVR elicits cellular responses in epidermal cells via several mechanisms: direct absorption of short-wavelength UVR photons by DNA, oxidative damage caused by long-wavelength UVR, and, as we recently demonstrated, via a retinal-dependent G protein-coupled signaling pathway. Because the human epidermis is exposed to a wide range of light wavelengths, we investigated whether opsins, light-activated receptors that mediate photoreception in the eye, are expressed in epidermal skin to potentially serve as photosensors. Here we show that four opsins—OPN1-SW, OPN2, OPN3 and OPN5—are expressed in the two major human epidermal cell types, melanocytes and keratinocytes, and the mRNA expression profile of these opsins does not change in response to physiological UVR doses. We detected two OPN3 splice variants present in similar amounts in both cell types and three OPN5 splice isoforms, two of which encode truncated proteins. Notably, OPN2 and OPN3 mRNA were significantly more abundant than other opsins and encoded full-length proteins. Our results demonstrate that opsins are expressed in epidermal skin cells and suggest that they might initiate light–induced signaling pathways, possibly contributing to UVR phototransduction. PMID:25267311

  3. Opsin expression in human epidermal skin.

    PubMed

    Haltaufderhyde, Kirk; Ozdeslik, Rana N; Wicks, Nadine L; Najera, Julia A; Oancea, Elena

    2015-01-01

    Human skin is constantly exposed to solar light containing visible and ultraviolet radiation (UVR), a powerful skin carcinogen. UVR elicits cellular responses in epidermal cells via several mechanisms: direct absorption of short-wavelength UVR photons by DNA, oxidative damage caused by long-wavelength UVR, and, as we recently demonstrated, via a retinal-dependent G protein-coupled signaling pathway. Because the human epidermis is exposed to a wide range of light wavelengths, we investigated whether opsins, light-activated receptors that mediate photoreception in the eye, are expressed in epidermal skin to potentially serve as photosensors. Here we show that four opsins—OPN1-SW, OPN2, OPN3 and OPN5—are expressed in the two major human epidermal cell types, melanocytes and keratinocytes, and the mRNA expression profile of these opsins does not change in response to physiological UVR doses. We detected two OPN3 splice variants present in similar amounts in both cell types and three OPN5 splice isoforms, two of which encode truncated proteins. Notably, OPN2 and OPN3 mRNA were significantly more abundant than other opsins and encoded full-length proteins. Our results demonstrate that opsins are expressed in epidermal skin cells and suggest that they might initiate light-induced signaling pathways, possibly contributing to UVR phototransduction. © 2014 The Authors. Photochemistry and Photobiology published by Wiley Periodicals, Inc. on behalf of American Society for Photobiology.

  4. Differential Gene Expression in Human Cerebrovascular Malformations

    PubMed Central

    Shenkar, Robert; Elliott, J. Paul; Diener, Katrina; Gault, Judith; Hu, Ling-Jia; Cohrs, Randall J.; Phang, Tzulip; Hunter, Lawrence; Breeze, Robert E.; Awad, Issam A.

    2009-01-01

    OBJECTIVE We sought to identify genes with differential expression in cerebral cavernous malformations (CCMs), arteriovenous malformations (AVMs), and control superficial temporal arteries (STAs) and to confirm differential expression of genes previously implicated in the pathobiology of these lesions. METHODS Total ribonucleic acid was isolated from four CCM, four AVM, and three STA surgical specimens and used to quantify lesion-specific messenger ribonucleic acid expression levels on human gene arrays. Data were analyzed with the use of two separate methodologies: gene discovery and confirmation analysis. RESULTS The gene discovery method identified 42 genes that were significantly up-regulated and 36 genes that were significantly down-regulated in CCMs as compared with AVMs and STAs (P = 0.006). Similarly, 48 genes were significantly up-regulated and 59 genes were significantly down-regulated in AVMs as compared with CCMs and STAs (P = 0.006). The confirmation analysis showed significant differential expression (P < 0.05) in 11 of 15 genes (angiogenesis factors, receptors, and structural proteins) that previously had been reported to be expressed differentially in CCMs and AVMs in immunohistochemical analysis. CONCLUSION We identify numerous genes that are differentially expressed in CCMs and AVMs and correlate expression with the immunohistochemistry of genes implicated in cerebrovascular malformations. In future efforts, we will aim to confirm candidate genes specifically related to the pathobiology of cerebrovascular malformations and determine their biological systems and mechanistic relevance. PMID:12535382

  5. Aquaporin expression in the human intervertebral disc.

    PubMed

    Richardson, S M; Knowles, R; Marples, D; Hoyland, J A; Mobasheri, A

    2008-06-01

    The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.

  6. Circadian Kisspeptin expression in human term placenta.

    PubMed

    de Pedro, M A; Morán, J; Díaz, I; Murias, L; Fernández-Plaza, C; González, C; Díaz, E

    2015-11-01

    Kisspeptin is an essential gatekeeper of reproductive function. During pregnancy high circulating levels of kisspeptin have been described, however the clear role of this neuropeptide in pregnancy remains unknown. We tested the existence of rhythmic kisspeptin expression in human full-term placenta from healthy pregnant women at six different time points during the day. The data obtained by Western blotting were fitted to a mathematical model (Fourier series), demonstrating, for the first time, the existence of a circadian rhythm in placental kisspeptin expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells.

    PubMed

    Rehnitz, Julia; Alcoba, Diego D; Brum, Ilma S; Hinderhofer, Katrin; Youness, Berthe; Strowitzki, Thomas; Vogt, Peter H

    2017-08-04

    Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  8. Expression of human. alpha. -fetoprotein in yeast

    SciTech Connect

    Yamamoto, Ritsu; Sakamoto, Takashi; Nishi, Shinzo; Sakai, Masaharu; Morinaga, Tomonori; Tamaoki, Taiki Univ. of Calgary, Alberta )

    1990-01-01

    Human {alpha}-fetoprotein (AFP) was expressed in Saccharomyces cerevisiae, with a plasmid containing the cDNA sequence for human AFP fused with the rat AFP signal peptide. The recombinant AFP was purified from the yeast lysate by DEAE-cellulose and immunoaffinity chromatography. The amino acid composition and the molecular weight of the recombinant AFP were similar to those of hepatoma AFP. N-terminal amino acids sequence analysis indicated that the signal peptide had been processed. The recombinant and hepatoma AFP reacted identically in Ouchterlony immunodiffusion and radioimmunoassay tests. These observations indicated that the yeast recombinant protein had the properties of native AFP.

  9. Effects and Interactions of Tachykinins and Dynorphin on FSH and LH Secretion in Developing and Adult Rats

    PubMed Central

    Ruiz-Pino, F.; Garcia-Galiano, D.; Manfredi-Lozano, M.; Leon, S.; Sánchez-Garrido, M. A.; Roa, J.; Pinilla, L.

    2015-01-01

    Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the

  10. Effects and interactions of tachykinins and dynorphin on FSH and LH secretion in developing and adult rats.

    PubMed

    Ruiz-Pino, F; Garcia-Galiano, D; Manfredi-Lozano, M; Leon, S; Sánchez-Garrido, M A; Roa, J; Pinilla, L; Navarro, V M; Tena-Sempere, M

    2015-02-01

    Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the

  11. Glycoprotein expression in human milk during lactation.

    PubMed

    Froehlich, John W; Dodds, Eric D; Barboza, Mariana; McJimpsey, Erica L; Seipert, Richard R; Francis, Jimi; An, Hyun Joo; Freeman, Samara; German, J Bruce; Lebrilla, Carlito B

    2010-05-26

    While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

  12. Tryptophan hydroxylase expression in human skin cells.

    PubMed

    Slominski, Andrzej; Pisarchik, Alexander; Johansson, Olle; Jing, Chen; Semak, Igor; Slugocki, George; Wortsman, Jacobo

    2003-10-15

    We attempted to further characterize cutaneous serotoninergic and melatoninergic pathways evaluating the key biosynthetic enzyme tryptophan hydroxylase (TPH). There was wide expression of TPH mRNA in whole human skin, cultured melanocytes and melanoma cells, dermal fibroblasts, squamous cell carcinoma cells and keratinocytes. Gene expression was associated with detection of TPH immunoreactive species by Western blotting. Characterization of the TPH immunoreactive species performed with two different antibodies showed expression of the expected protein (55-60 kDa), and of forms with higher and lower molecular weights. This pattern of broad spectrum of TPH expression including presumed degradation products suggests rapid turnover of the enzyme, as previously reported in mastocytoma cells. RP-HPLC of skin extracts showed fluorescent species with the retention time of serotonin and N-acetylserotonin. Immunocytochemistry performed in skin biopsies localized TPH immunoreactivity to normal and malignant melanocytes. We conclude that while the TPH mRNA and protein are widely expressed in cultured normal and pathological epidermal and dermal skin cells, in vivo TPH expression is predominantly restricted to cells of melanocytic origin.

  13. FOXO1/3 Depletion in Granulosa Cells Alters Follicle Growth, Death and Regulation of Pituitary FSH

    PubMed Central

    Liu, Zhilin; Castrillon, Diego H.; Zhou, Wei

    2013-01-01

    The Forkhead boxO (FOXO) transcription factors regulate multiple cellular functions. FOXO1 and FOXO3 are highly expressed in granulosa cells of ovarian follicles. Selective depletion of the Foxo1 and Foxo3 genes in granulosa cells of mice reveals a novel ovarian-pituitary endocrine feedback loop characterized by: 1) undetectable levels of serum FSH but not LH, 2) reduced expression of the pituitary Fshb gene and its transcriptional regulators, and 3) ovarian production of a factor(s) that suppresses pituitary cell Fshb expression. Equally notable, and independent of FSH, microarray analyses and quantitative PCR document that depletion of Foxo1/3 alters the expression of specific genes associated with follicle growth vs. apoptosis by disrupting critical and selective regulatory interactions of FOXO1/3 with the activin or bone morphogenetic protein 2 (BMP2) pathways, respectively. As a consequence, both granulosa cell proliferation and apoptosis were decreased. These data provide the first evidence that FOXO1/3 divergently regulate follicle growth or death by interacting with the activin or BMP pathways in granulosa cells and by modulating pituitary FSH production. PMID:23322722

  14. FOXO1/3 depletion in granulosa cells alters follicle growth, death and regulation of pituitary FSH.

    PubMed

    Liu, Zhilin; Castrillon, Diego H; Zhou, Wei; Richards, Joanne S

    2013-02-01

    The Forkhead boxO (FOXO) transcription factors regulate multiple cellular functions. FOXO1 and FOXO3 are highly expressed in granulosa cells of ovarian follicles. Selective depletion of the Foxo1 and Foxo3 genes in granulosa cells of mice reveals a novel ovarian-pituitary endocrine feedback loop characterized by: 1) undetectable levels of serum FSH but not LH, 2) reduced expression of the pituitary Fshb gene and its transcriptional regulators, and 3) ovarian production of a factor(s) that suppresses pituitary cell Fshb expression. Equally notable, and independent of FSH, microarray analyses and quantitative PCR document that depletion of Foxo1/3 alters the expression of specific genes associated with follicle growth vs. apoptosis by disrupting critical and selective regulatory interactions of FOXO1/3 with the activin or bone morphogenetic protein 2 (BMP2) pathways, respectively. As a consequence, both granulosa cell proliferation and apoptosis were decreased. These data provide the first evidence that FOXO1/3 divergently regulate follicle growth or death by interacting with the activin or BMP pathways in granulosa cells and by modulating pituitary FSH production.

  15. Genes of periodontopathogens expressed during human disease.

    PubMed

    Song, Yo-Han; Kozarov, Emil V; Walters, Sheila M; Cao, Sam Linsen; Handfield, Martin; Hillman, Jeffrey D; Progulske-Fox, Ann

    2002-12-01

    Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.

  16. Emotion expression in human punishment behavior.

    PubMed

    Xiao, Erte; Houser, Daniel

    2005-05-17

    Evolutionary theory reveals that punishment is effective in promoting cooperation and maintaining social norms. Although it is accepted that emotions are connected to punishment decisions, there remains substantial debate over why humans use costly punishment. Here we show experimentally that constraints on emotion expression can increase the use of costly punishment. We report data from ultimatum games, where a proposer offers a division of a sum of money and a responder decides whether to accept the split, or reject and leave both players with nothing. Compared with the treatment in which expressing emotions directly to proposers is prohibited, rejection of unfair offers is significantly less frequent when responders can convey their feelings to the proposer concurrently with their decisions. These data support the view that costly punishment might itself be used to express negative emotions and suggest that future studies will benefit by recognizing that human demand for emotion expression can have significant behavioral consequences in social environments, including families, courts, companies, and markets.

  17. A Comparison of Artificial Subtle Expressions with Human-like Expressions on Expressing Confidence Level

    NASA Astrophysics Data System (ADS)

    Komatsu, Takanori; Kobayashi, Kazuki; Yamada, Seiji; Funakoshi, Kotaro; Nakano, Mikio

    Expressing the confidence level of a system's suggestions by using speech sounds is an important cue to users of the system for perceiving how likely it is for the suggestions to be correct. We assume that expressing confidence levels by using human-like expressions would cause users to have a poorer impression of the systems than if artificial subtle expressions (ASEs) were used when the quality of the presented information does not match the expressed confidence level. We confirmed that this assumption was correct by conducting a psychological experiment.

  18. HP-HMG versus rFSH in treatments combining fresh and frozen IVF cycles: success rates and economic evaluation.

    PubMed

    Wex-Wechowski, Jaro; Abou-Setta, Ahmed M; Kildegaard Nielsen, Sandy; Kennedy, Richard

    2010-08-01

    The economic implications of the choice of gonadotrophin influence decision making but their cost-effectiveness in frozen-embryo transfer cycles has not been adequately studied. An economic evaluation was performed comparing highly purified human menopausal gonadotrophin (HP-HMG) and recombinant FSH (rFSH) using individual patient data (n=986) from two large randomized controlled trials using a long agonist IVF protocol. The simulation model incorporated live birth data and published UK costs of IVF-related medical resources. After treatment for up-to-three cycles (one fresh and up to two subsequent fresh or frozen cycles conditional on availability of cryopreserved embryos), the cumulative live birth rate was 53.7% (95% CI 49.3-58.1%) for HP-HMG and 44.6% (40.2-49.0%) for rFSH (OR 1.44, 95% CI 1.12-1.85; P<0.005). The mean costs per IVF treatment for HP-HMG and rFSH were pound5393 ( pound5341-5449) and pound6269 ( pound6210-6324), respectively (number needed to treat to fund one additional treatment was seven; P<0.001). With maternal and neonatal costs applied, the median cost per IVF baby delivered with HP-HMG was pound11,157 ( pound11,089-11,129) and pound14,227 ( pound14,183-14,222) with rFSH (P<0.001). The cost saving using HP-HMG remained after varying model parameters in a probabilistic sensitivity analysis.

  19. Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

    SciTech Connect

    Smith, R.A.; Branca, A.A.; Reichert, L.E. Jr.

    1985-11-15

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs.

  20. Antifolliculogenic action of progesterone despite hypersecretion of FSH in monkeys.

    PubMed

    Goodman, A L; Hodgen, G D

    1982-11-01

    To learn how progesterone (P) inhibits follicle growth during the luteal phase, we determined whether P will inhibit follicle growth when follicle-stimulating hormone (FSH) is secreted in large amounts, namely, after luteectomy (CLX) in monkeys with only one ovary. Second, a functional role for 17 alpha-hydroxyprogesterone (17OHP) was examined as a common mediator of the inhibition of folliculogenesis by the dominant follicle and corpus luteum. To accomplish the first goal, nine chronically hemiovarectomized monkeys were lutectomized chronically hemiovariectomized monkeys were luteectomized at midluteal phase. In five monkeys that received no steroid, the next preovulatory luteinizing hormone (LH) surge occurred 14.0 +/- 0.8 days (mean +/- SE) after CLX. In contrast, the next LH surge was delayed in four monkeys implanted for 10 days with Silastic capsules containing P and occurred 25.0 +/- 2.7 days after CLX, i.e., 14.8 +/- 2.7 days after the capsule removal. In both groups, FSH levels increased markedly after CLX to a comparable degree and duration; yet, only a single follicle ovulated in each monkey. To examine a potential inhibitory role for 17OHP, monkeys with two ovaries were luteectomized and received 1) no steroid, 2) 17OHP via Silastic capsules, or 3) P for 10 days after CLX. Progesterone replacement after CLX appeared to maintain 17OHP levels, which showed a transient decrease after CLX alone. As above, P delayed the next LH surge (25.4 +/- 1.3 vs. 15.0 +/- 0.6 days) despite comparable increases in serum FSH after CLX alone. Replacement at two levels of 17OHP did not delay the onset of menses (2-3 days post-CLX) or significantly delay the next LH surge 18.3 +/!- 1.9 or 20.8 +/- 3.4 vs. 15.0 +/- 0.6 days (P greater than 0.2) in monkeys CLX only. Whatever may be the mode of action of P, it appears that it is not mediated by peripheral conversion to 17OHP. These findings demonstrate that P at luteal phase levels can inhibit follicle growth culminating in

  1. The measurement of LH, FSH, and prolactin.

    PubMed

    Wheeler, Michael J

    2013-01-01

    In the clinical laboratory, the reproductive hormones are probably the second most commonly measured hormones after the thyroid hormones. More than 300 laboratories participate in the UK National External Quality Control Scheme. In addition, investigations into reproduction and fertility in humans and animals remain a major area of research. Illustrative methods are described for the three reproductive hormones (luteinizing hormone, follicle-stimulating hormone, prolactin). Radioimmunoassay, immunoradiometric, and enzyme assays are described to give a wide choice of assay formats. There are many commercial assays available and illustrative ones are described. Possible interferences are discussed and procedures for investigating their presence and removal are given.

  2. Anti-FSH antibodies associate with poor outcome of ovarian stimulation in IVF.

    PubMed

    Haller, Kadri; Salumets, Andres; Uibo, Raivo

    2008-03-01

    FSH is required for spontaneous folliculogenesis and is widely used in ovarian stimulation in IVF. Previously, increased concentrations of antibodies against FSH (anti-FSH) have been demonstrated in infertile women. This study aimed to: (i) assess the possible association of anti-FSH with an adverse outcome of IVF with regard to clinical parameters characterizing the ovarian reserve; and (ii) compare serum and follicular fluid (FF) anti-FSH concentrations in relation to follicle size and endocrine markers. IVF patients (n = 182) subjected to gonadotrophin-releasing hormone-antagonist protocol were assessed for anti-FSH using enzyme-linked immunosorbent assay. Increased concentrations of serum anti-FSH immunoglobulin (Ig)G and IgA were associated with impaired ovarian stimulation outcome, with cut-off values <1.0 arbitrary units predicting poor ovarian response (FSH IgG and IgA were positively associated with serum anti-FSH concentrations and FSH concentration in FF. Additionally, FF anti-FSH IgG increased with follicle growth (linear regression coefficient = 0.02, P = 0.022). Collectively, these data suggest that serum anti-FSH antibodies are associated with poor ovarian response to FSH stimulation in IVF, with anti-FSH IgA and IgG potentially exerting a local FSH antagonizing effect in maturing follicles.

  3. Gene expression profiling of human ovarian tumours

    PubMed Central

    Biade, S; Marinucci, M; Schick, J; Roberts, D; Workman, G; Sage, E H; O'Dwyer, P J; LiVolsi, V A; Johnson, S W

    2006-01-01

    There is currently a lack of reliable diagnostic and prognostic markers for ovarian cancer. We established gene expression profiles for 120 human ovarian tumours to identify determinants of histologic subtype, grade and degree of malignancy. Unsupervised cluster analysis of the most variable set of expression data resulted in three major tumour groups. One consisted predominantly of benign tumours, one contained mostly malignant tumours, and one was comprised of a mixture of borderline and malignant tumours. Using two supervised approaches, we identified a set of genes that distinguished the benign, borderline and malignant phenotypes. These algorithms were unable to establish profiles for histologic subtype or grade. To validate these findings, the expression of 21 candidate genes selected from these analyses was measured by quantitative RT–PCR using an independent set of tumour samples. Hierarchical clustering of these data resulted in two major groups, one benign and one malignant, with the borderline tumours interspersed between the two groups. These results indicate that borderline ovarian tumours may be classified as either benign or malignant, and that this classifier could be useful for predicting the clinical course of borderline tumours. Immunohistochemical analysis also demonstrated increased expression of CD24 antigen in malignant versus benign tumour tissue. The data that we have generated will contribute to a growing body of expression data that more accurately define the biologic and clinical characteristics of ovarian cancers. PMID:16969345

  4. Gene expression profiling of human ovarian tumours.

    PubMed

    Biade, S; Marinucci, M; Schick, J; Roberts, D; Workman, G; Sage, E H; O'Dwyer, P J; Livolsi, V A; Johnson, S W

    2006-10-23

    There is currently a lack of reliable diagnostic and prognostic markers for ovarian cancer. We established gene expression profiles for 120 human ovarian tumours to identify determinants of histologic subtype, grade and degree of malignancy. Unsupervised cluster analysis of the most variable set of expression data resulted in three major tumour groups. One consisted predominantly of benign tumours, one contained mostly malignant tumours, and one was comprised of a mixture of borderline and malignant tumours. Using two supervised approaches, we identified a set of genes that distinguished the benign, borderline and malignant phenotypes. These algorithms were unable to establish profiles for histologic subtype or grade. To validate these findings, the expression of 21 candidate genes selected from these analyses was measured by quantitative RT-PCR using an independent set of tumour samples. Hierarchical clustering of these data resulted in two major groups, one benign and one malignant, with the borderline tumours interspersed between the two groups. These results indicate that borderline ovarian tumours may be classified as either benign or malignant, and that this classifier could be useful for predicting the clinical course of borderline tumours. Immunohistochemical analysis also demonstrated increased expression of CD24 antigen in malignant versus benign tumour tissue. The data that we have generated will contribute to a growing body of expression data that more accurately define the biologic and clinical characteristics of ovarian cancers.

  5. Expression profiling the human septin gene family.

    PubMed

    Hall, Peter A; Jung, Kenneth; Hillan, Kenneth J; Russell, S E Hilary

    2005-07-01

    The septins are an evolutionarily conserved family of GTP-binding proteins involved in diverse processes including vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration, and neoplasia. The present paper reports a comprehensive study of septin gene expression by DNA microarray methods in 10 360 samples of normal, diseased, and tumour tissues. A novel septin, SEPT13, has been identified and is shown to be related to SEPT7. It is shown that SEPT13 and the other known human septins are expressed in all tissue types but some show high expression in lymphoid (SEPT1, 6, 9, and 12) or brain tissues (SEPT2, 3, 4, 5, 7, 8, and 11). For a given septin, some isoforms are highly expressed in the brain and others are not. For example, SEPT8_v2 and v1, 1* and 3 are highly expressed in the brain and cluster with SEPT2, 3, 4, 5, 7, and 11. However, a probe set specific for SEPT8_v1 with low brain expression clusters away from this set. Similarly, SEPT4 has lymphoid and non-lymphoid forms; SEPT2 has lymphoid and central nervous system (CNS) forms; and SEPT6 and SEPT9 are elevated in lymphoid tissues but both have forms that cluster away from the lymphoid forms. Perturbation of septin expression was widespread in disease and tumours of the various tissues examined, particularly for conditions of the CNS, where alterations in all 13 septin genes were identified. This analysis provides a comprehensive catalogue of the septin family in health and disease. It is a key step in understanding the role of septins in physiological and pathological states and provides insight into the complexity of septin biology. Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  6. Effects of different batches of /sup 125/iodine on properties of /sup 125/I-hFSH and characteristics of radioligand-receptor assays

    SciTech Connect

    Melson, B.E.; Sluss, P.M.; Reichert, L.E. Jr.

    1987-02-01

    Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCI carrier free Na/sup 125/I (Amersham Corp., 15 mCi/micrograms I2) in the presence of lactoperoxidase. Incorporation of /sup 125/I into hFSH was determined by the method of R. C. Greenwood, W. M. Hunter, and J. S. Grover (1963) Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20 degrees C) using different calf testis membrane preparations having similar receptor characteristics. Each /sup 125/I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of Ka and Rt. Incorporation of /sup 125/I into FSH was relatively constant over the large number of experiments (62.4 +/- 6.4 microCi/micrograms; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of /sup 125/I-hFSH was related to the lot of /sup 125/I utilized, and was significantly (P less than or equal to 0.01) lower and more variable (28.7 +/- 10.5 microCi/micrograms). Maximum bindability of /sup 125/I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH /sup 125/I incorporation (r = -0.47; P less than or equal to 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na/sup 125/I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.

  7. Relationship between serum gonadotropins and pituitary immunoreactive gonadotropins and steroid receptors during the first FSH increase of the estrous cycle and following steroid treatment in heifers.

    PubMed

    Lane, Elizabeth A; Sweeney, Torres; Ryan, Marion; Roche, James F; Crowe, Mark A

    2009-05-01

    The objectives were to determine the effects of (i) time during the first FSH increase of the estrous cycle (time-course study) and (ii) exogenous steroid treatment (steroid feedback study) on the relationship between circulating serum gonadotropins, and the proportions of pituitary cells immunoreactive for gonadotropins and steroid receptors during the estrous cycle in heifers. Pituitaries were collected from heifers (n=40) slaughtered at 13h (n=8), 30h (n=24) and 66h (n=8) after estrous onset, corresponding to before, during and after the first FSH increase of the estrous cycle. Heifers slaughtered during the FSH increase (at 30h) either received no treatment (n=8), or were treated (n=16) with estradiol benzoate and/or progesterone before slaughter. During the time-course study, the proportion of pituitary cells immunoreactive for FSH increased (P<0.05) during the first transient FSH increase reflecting serum concentrations. The proportion of pituitary cells immunoreactive for LH was unaltered, a reflection of serum LH concentrations. The proportion of estrogen receptors (ER)-alpha, but not ER-beta, was decreased (P<0.05) at 30h compared with at either 13 or 66h. During the steroid feedback study, exogenous progesterone with or without estradiol suppressed (P<0.05) the proportions of pituitary cells immunoreactive for gonadotropins, serum FSH concentrations and LH pulse frequency. Steroid treatment did not alter the proportion of pituitary cells positive for estrogen receptors (alpha and beta). While progesterone receptors (PR) were not detected in the anterior pituitary by immunohistochemistry during the early estrous cycle or in response to steroid treatment, quantitative real-time PCR revealed that mRNA for progesterone receptors was expressed at very low levels. The expression of pituitary PR mRNA was decreased (P<0.05) at 30 and 66h compared with 13h, and was suppressed (P<0.05) following steroid treatments. Alterations in pituitary steroid receptors are

  8. Testicular enlargement in a patient with a FSH-secreting pituitary adenoma.

    PubMed

    Dahlqvist, Per; Koskinen, Lars-Owe D; Brännström, Thomas; Hägg, Erik

    2010-04-01

    Clinically non-functional pituitary adenomas are often derived from gonadotropin producing cells. However, gonadotropinomas causing elevated serum levels of follicle-stimulating hormone (FSH) and clinical signs of FSH hypersecretion are very rarely described. Our patient, a 56-year-old man, was referred to our clinic with signs of hypogonadism. Magnetic resonance imaging (MRI) and biochemical examinations showed a large pituitary adenoma and excessive levels of serum FSH. Clinical examination and ultrasound measurement revealed bilaterally enlarged testes. After pituitary surgery, serum FSH levels normalized and there was a decrease in testicular volume. This case suggests that supraphysiological levels of FSH from a gonadotropinoma can cause a clinically observable effect, i.e. testicular enlargement. This is in line with experimental studies showing biological effect of FSH from pituitary adenomas and previous occasional reports of ovarian hyperstimulation and testicular enlargement in patients with FSH-secreting gonadotropinomas.

  9. Hormone Receptor Expression in Human Fascial Tissue

    PubMed Central

    Fede, C.; Albertin, G.; Petrelli, L.; Sfriso, M.M.; Biz, C.; De Caro, R.

    2016-01-01

    Many epidemiologic, clinical, and experimental findings point to sex differences in myofascial pain in view of the fact that adult women tend to have more myofascial problems with respect to men. It is possible that one of the stimuli to sensitization of fascial nociceptors could come from hormonal factors such as estrogen and relaxin, that are involved in extracellular matrix and collagen remodeling and thus contribute to functions of myofascial tissue. Immunohistochemical and molecular investigations (real-time PCR analysis) of relaxin receptor 1 (RXFP1) and estrogen receptor-alpha (ERα) localization were carried out on samples of human fascia collected from 8 volunteers patients during orthopedic surgery (all females, between 42 and 70 yrs, divided into pre- and post-menopausal groups), and in fibroblasts isolated from deep fascia, to examine both protein and RNA expression levels. We can assume that the two sex hormone receptors analyzed are expressed in all the human fascial districts examined and in fascial fibroblasts culture cells, to a lesser degree in the post-menopausal with respect to the pre-menopausal women. Hormone receptor expression was concentrated in the fibroblasts, and RXFP1 was also evident in blood vessels and nerves. Our results are the first demonstrating that the fibroblasts located within different districts of the muscular fasciae express sex hormone receptors and can help to explain the link between hormonal factors and myofascial pain. It is known, in fact, that estrogen and relaxin play a key role in extracellular matrix remodeling by inhibiting fibrosis and inflammatory activities, both important factors affecting fascial stiffness and sensitization of fascial nociceptors. PMID:28076930

  10. Medical humanities as expressive of Western culture.

    PubMed

    Hooker, Claire; Noonan, Estelle

    2011-12-01

    In this paper we articulate a growing awareness within the field of the ways in which medical humanities could be deemed expressive of Western cultural values. The authors suggest that medical humanities is culturally limited by a pedagogical and scholarly emphasis on Western cultural artefacts, as well as a tendency to enact an uncritical reliance upon foundational concepts (such as 'patient' and 'experience') within Western medicine. Both these tendencies within the field, we suggest, are underpinned by a humanistic emphasis on appreciative or receptive encounters with 'difference' among patients that may unwittingly contribute to the marginalisation of some patients and healthcare workers. While cultural difference should be acknowledged as a central preoccupation of medical humanities, we argue that the discipline must continue to expand its scholarly and critical engagements with processes of Othering in biomedicine. We suggest that such improvements are necessary in order to reflect the cultural diversification of medical humanities students, and the geographical expansion of the discipline within non-Western and/or non-Anglophone locations.

  11. Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

    PubMed

    Liu, Jianqi; Kuulasmaa, Tiina; Kosma, Veli-Matti; Bützow, Ralf; Vänttinen, Teemu; Hydén-Granskog, Christel; Voutilainen, Raimo

    2003-10-01

    Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to

  12. Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging.

    PubMed

    Khan, Muhammad Irfan-Ur-Rehman; Dias, Fernanda Caminha Faustino; Dufort, Isabelle; Misra, Vikram; Sirard, Marc-Andre; Singh, Jaswant

    2016-04-01

    The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P>0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P=0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.

  13. Effects of leptin on FSH cells in the pituitary gland of Podarcis siculus.

    PubMed

    Ferrandino, Ida; Monaco, Antonio; Grimaldi, Maria Consiglio

    2015-03-01

    Leptin is the hormone synthesised by adipocytes, which plays an important role in regulating appetite and metabolism. In mammals, this pleiotropic hormone also plays a key role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions. However, little is known about leptin in lower vertebrates and particularly few studies are available on reptiles. In the present work, we analysed the action of recombinant human leptin on FSH cells in the pituitary gland of Podarcis siculus female lizards exposed to four different concentrations of the hormone. FSH cells showed a dose-dependent reaction. The data are indicative of the role played by leptin in modulating the cellular activity of such cells in the pituitary gland of P. siculus, similar to what was already reported in mammals. A functional receptor is evidently able to respond to leptin in this lizard, but further comparative studies are needed to understand the role of this hormone in ectothermic vertebrates. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  14. Induction of spermatogenesis by rhFSH for azoospermia due to spermatogenic dysfunction with maturation arrest: five case series.

    PubMed

    Kobori, Yoshitomo; Suzuki, Keisuke; Iwahata, Toshiyuki; Shin, Takeshi; Sato, Ryo; Nishio, Kojiro; Yagi, Hiroshi; Arai, Gaku; Soh, Shigehiro; Okada, Hiroshi

    2015-06-01

    When sperm cannot be retrieved from the testes of patients with azoospermia due to spermatogenic dysfunction (ASD), there is no rational way for the patient to become a biological father. We investigated the possibility of inducing spermatogenesis in such patients by hormonal therapy with recombinant human follicle-stimulating hormone (rhFSH) alone. Twenty-six ASD patients who could not obtain spermatozoa by microdissection testicular sperm extraction (micro-TESE) were confirmed to have arrested spermatogenesis at the late stage of maturation arrest. They were subsequently treated with 75-150 IU two times/week rhFSH alone for 12 months. The primary endpoint was the appearance of sperm in ejaculate, and we followed the patients to determine the outcome of inseminating their partners. After rhFSH treatment, mature spermatozoa were found in the ejaculate in five of 26 (19.2%) patients, all of whom showed histology of non-uniform type maturation arrest. Intracytoplasmic sperm injection of the mature spermatozoa resulted in two ongoing clinical pregnancies (insemination success rate, 40.0%). Recombinant human follicle-stimulating hormone treatment can be used as an advanced assisted reproductive technology to improve spermatogenesis in some azoospermic patients with maturation arrest of spermatogenesis and is a potential treatment option after unsuccessful micro-TESE.

  15. Expression of Cellular Oncogenes in Human Malignancies

    NASA Astrophysics Data System (ADS)

    Slamon, Dennis J.; Dekernion, Jean B.; Verma, Inder M.; Cline, Martin J.

    1984-04-01

    Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

  16. Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase expression and activity in the hypophysectomized rat ovary: Interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin

    SciTech Connect

    Martel, C.; Labrie, C.; Dupont, E.; Couet, J.; Trudel, C.; Rheaume, E.; Simard, J.; Luu-The, V.; Pelletier, G.; Labrie, F. )

    1990-12-01

    The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes an obligatory step in the conversion of pregnenolone and other 5-ene-3 beta-hydroxysteroids into progesterone as well as precursors of all androgens and estrogens in the ovary. Since 3 beta-HSD is likely to be an important target for regulation by pituitary hormones, we have studied the effect of chronic treatment with LH (hCG), FSH, and PRL on ovarian 3 beta-HSD expression and activity in hypophysectomized adult female rats. Human CG (hCG) (10 IU, twice a day (bid)), ovine FSH (0.5 microgram, bid), and ovine PRL (1 mg, bid) were administered, singly or in combination, for a period of 10 days starting 15 days after hypophysectomy. In hypophysectomized rats, PRL exerted a potent inhibitory effect on all the parameters studied. In fact, PRL caused a 81% decrease in ovarian 3 beta-HSD mRNA content accompanied by a similar decrease in 3 beta-HSD activity and protein levels. In addition, ovarian weight decreased by 40% whereas serum progesterone fell dramatically from 1.92 nmol/liter to undetectable levels after treatment with PRL. Whereas hCG alone had only slight stimulatory effects on 3 beta-HSD mRNA, protein content and activity levels, treatment with the gonadotropin partially or completely reversed the potent inhibitory effects of oPRL on all the parameters measured. FSH, on the other hand, had no significant effect on 3 beta-HSD expression and activity. In situ hybridization experiments using the 35S-labeled rat ovary 3 beta-HSD cDNA probe show that the inhibitory effect of PRL is exerted primarily on luteal cell 3 beta-HSD expression and activity. On the other hand, it can be seen that hCG stimulates 3 beta-HSD mRNA accumulation in interstitial cells.

  17. A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice.

    PubMed

    Wang, Huizhen; Butnev, Vladimir; Bousfield, George R; Kumar, T Rajendra

    2016-05-05

    Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are

  18. GLUT-3 expression in human skeletal muscle

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

    2000-01-01

    Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

  19. GLUT-3 expression in human skeletal muscle

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

    2000-01-01

    Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

  20. The pharmacology of human appetite expression.

    PubMed

    Halford, Jason C G; Cooper, Gillian D; Dovey, Terence M

    2004-04-01

    The discovery of the adiposity signal leptin a decade ago revolutionised our understanding of the hypothalamic mechanisms underpinning the central control of ingestive behaviour. Subsequently, the structure and function of various hypothalamic peptide systems (Neuropeptide Y (NPY), Orexins, Melanocortins, Cocaine and Amphetamine Regulating Transcript (CART), Galanin/Galanin Like Peptides (GALP) and endocannabinoids) have been characterised in detail in rodent models. The therapeutic benefit of targeting these systems remains to be discovered. More is becoming known about the pharmacological potential of peripheral, meal-induced, episodic endogenous peptides. Hormones such as Cholecystokinin (CCK), Gastrin Releasing Peptides (GRP), Glucagon-Like Peptide I (GLP-1) Enterostatin, Amylin, Peptide YY (PYY) and Ghrelin are released prior to, during and/or after a meal, controlling intake and subjective feelings of appetite (hunger and satiety). In addition, there is an expanding body of literature detailing the effects of a wide variety of drugs on human appetite and food intake. Some of these drugs act upon CNS monoamine systems such as Serotonin (5-HT). Dopamine (DA) and Noradrenaline (NA), have long been implicated in appetite regulation. Detailed examination of both the effect of agonising endogenous gut peptide systems and the effect of various monoaminergic drugs on the expression of human appetite can provide a greater understanding of mechanisms underpinning normal appetite regulation. However, such an understanding must be based on knowledge of the effect of the treatment on meal size, eating rate, meal pattern, food choice and the subjective experience of appetite flux (hunger and satiety), and notjust food intake.

  1. Effects of ovum pick-up frequency and FSH stimulation: a retrospective study on seven years of beef cattle in vitro embryo production.

    PubMed

    De Roover, R; Feugang, J M N; Bols, P E J; Genicot, G; Hanzen, Ch

    2008-04-01

    The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non-stimulated Ovum Pick-up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH-LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day -8). The dominant follicle was ablated by ultrasound-guided follicle puncture on day -6. On day -3 and day -2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm), i.e. a total dose of 160 mug FSH and 40 mug LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14-day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set-up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2-week periods are considered (four non-stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non-stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better-quality, oocytes.

  2. Expression of CD4 by human megakaryocytes.

    PubMed Central

    Basch, R S; Kouri, Y H; Karpatkin, S

    1990-01-01

    The CD4 antigen, which serves as the receptor for human immunodeficiency virus type 1 (HIV-1) on T cells, has been detected on human megakaryocytes. Recent evidence of impaired thrombopoiesis in HIV-1-related thrombocytopenia suggested that these cells could be directly infected by the virus and prompted a search for a receptor on megakaryocytes of normal subjects that could permit entry of HIV-1. Bone marrow specimens from uninfected normal control subjects were centrifuged over Ficoll-Hypaque (1.077 g/ml) and analyzed by three-color analysis with a flow cytometer utilizing monoclonal antibodies against CD4 and a glycoprotein present on the surface of megakaryocytes and platelets (GPIIb/IIIa; CD41), as well as 7-aminoactinomycin D, a stain for DNA. Cells presumed to be megakaryocytes were identified by having a DNA content greater than tetraploid and staining brightly with anti-CD41. Approximately 0.4% of the nucleated cells of the marrow met these criteria. Twenty-five percent of these megakaryocytes stained as brightly as CD4+ T cells. Several clones of antibody recognizing different epitopes of the CD4 molecule gave similar results. Platelets were CD4-. Staining of megakaryocytes with anti-CD4 was confirmed by direct microscopic examination of Percoll-gradient-enriched megakaryocytes employing two-color (CD4-phycoerythrin and CD41-fluorescein) immunofluorescence analysis and phase-contrast microscopy. The proportion of double-labeled cells among 112 phase-contrast-identifiable megakaryocytes from five bone marrow specimens varied between 20% and 26% with a mean and SD of 22% +/- 2.5%. Thus some human megakaryocytes express CD4 on their surface that should be capable of binding the HIV-1 gp120 envelope protein. This could serve as a portal of entry for HIV-1. Images PMID:2236021

  3. Heterogeneity of rat FSH by chromatofocusing: studies on in-vitro bioactivity of pituitary FSH forms and effect of neuraminidase treatment.

    PubMed

    Blum, W F; Riegelbauer, G; Gupta, D

    1985-04-01

    This study concerned the resolution of rat pituitary FSH utilizing chromatofocusing. Among the 11 components resolved and positively identified, ten had apparent isoelectric points (pI) between 3.1 and 5.1. Approximately 1% of pituitary FSH eluted at pH 9.4. Treatment with varying amounts of neuraminidase followed by refocusing generated FSH components of higher pI values. Treatment with other glycosidases did not alter the elution characteristics in chromatofocusing, while exclusion chromatography established an inverse relationship between apparent molecular weight and pI. Dose-response curves of various FSH components and of the reference preparation in the current radioimmunoassay system were parallel to each other. A study of their in-vitro bioactivity, utilizing granulosa cells which produce a plasminogen activator due to FSH in a dose-dependent manner, provided the following evidence: increased acidity of the components led to an increase of maximum response and an increase of the dose necessary for half-maximum response. Considering the observed alterations in the heterogeneity of FSH with changing physiological states of the animal, it is concluded that qualitative changes of the FSH molecule are perhaps involved in a modulatory role in the biopotencies of the hormone.

  4. Age-specific reference values for serum FSH and estradiol levels throughout the reproductive period.

    PubMed

    Grisendi, Valentina; Spada, Elena; Argento, Cindy; Plebani, Maddalena; Milani, Silvano; Seracchioli, Renato; Volpe, Annibale; La Marca, Antonio

    2014-06-01

    High serum day 3 FSH levels are associated with poor ovarian reserve and reduced fertility, but the interpretation of FSH values according to age is still not univocal. The purpose of this study was to determine age-dependent reference values in women with regular menstrual cycles and FSH as a guide for specialists. The study was performed at the Department of Mother-Infant of a University-based tertiary care centre. One-hundred ninety-two healthy normal menstruating women were recruited for the study. All patients attended the department on menstrual cycle day 3 for a blood sample for FSH and estradiol determination. A linear relationship between FSH or estradiol serum levels and age was observed. The FSH level increased by 0.11 IU for every year of age (1 IU for every 9 years of age). The values of FSH and estradiol corresponding to the 5th, 25th, 50th, 75th, 95th centiles for any specific age have been calculated. Serum FSH levels need to be interpreted according to age-dependent reference values. Serum FSH levels on 95th centile for any age may represent a warning sign for reduced ovarian reserve.

  5. Individualization of the FSH starting dose in IVF/ICSI cycles using the antral follicle count

    PubMed Central

    2013-01-01

    Background The FSH starting dose is usually chosen according to women’s age, anamnesis, clinical criteria and markers of ovarian reserve. Currently used markers include antral follicle count (AFC), which is considered to have a very high performance in predicting ovarian response to FSH. The objective of the present study to elaborate a nomogram based on AFC for the calculation of the appropriate FSH starting dose in IVF cycles. Methods This is a retrospective study performed at the Mother-Infant Department of Modena University Hospital. IVF patients (n=505) were subjected to blood sampling and transvaginal ultrasound for measurement of serum day3 FSH, estradiol and AFC. The variables predictive of the number of retrieved oocytes were assessed by backwards stepwise multiple regression. The variables reaching the statistical significance were then used in the calculation for the final predictive model. Results A model based on age, AFC and FSH was able to accurately predict the ovarian sensitivity and accounted for 30% of the variability of ovarian response to FSH. An FSH dosage nomogram was constructed and overall it predicts a starting dose lower than 225 IU in 50.2% and 18.1% of patients younger and older than 35 years, respectively. Conclusions The daily FSH dose may be calculated on the basis of age and two markers of ovarian reserve, namely AFC and FSH, with the last two variables being the most significant predictors. The nomogram seems easily applicable during the daily clinical practice. PMID:23388048

  6. Individualization of the FSH starting dose in IVF/ICSI cycles using the antral follicle count.

    PubMed

    La Marca, Antonio; Grisendi, Valentina; Giulini, Simone; Argento, Cindy; Tirelli, Alessandra; Dondi, Giulia; Papaleo, Enrico; Volpe, Annibale

    2013-02-06

    The FSH starting dose is usually chosen according to women's age, anamnesis, clinical criteria and markers of ovarian reserve. Currently used markers include antral follicle count (AFC), which is considered to have a very high performance in predicting ovarian response to FSH. The objective of the present study to elaborate a nomogram based on AFC for the calculation of the appropriate FSH starting dose in IVF cycles. This is a retrospective study performed at the Mother-Infant Department of Modena University Hospital. IVF patients (n=505) were subjected to blood sampling and transvaginal ultrasound for measurement of serum day3 FSH, estradiol and AFC. The variables predictive of the number of retrieved oocytes were assessed by backwards stepwise multiple regression. The variables reaching the statistical significance were then used in the calculation for the final predictive model. A model based on age, AFC and FSH was able to accurately predict the ovarian sensitivity and accounted for 30% of the variability of ovarian response to FSH. An FSH dosage nomogram was constructed and overall it predicts a starting dose lower than 225 IU in 50.2% and 18.1% of patients younger and older than 35 years, respectively. The daily FSH dose may be calculated on the basis of age and two markers of ovarian reserve, namely AFC and FSH, with the last two variables being the most significant predictors. The nomogram seems easily applicable during the daily clinical practice.

  7. Endocrine control of spermatogenesis: Role of FSH and LH/ testosterone

    PubMed Central

    Ramaswamy, Suresh; Weinbauer, Gerhard F

    2014-01-01

    Evaluation of testicular functions (production of sperm and androgens) is an important aspect of preclinical safety assessment and testicular toxicity is comparatively far more common than ovarian toxicity. This chapter focuses (1) on the histological sequelae of disturbed reproductive endocrinology in rat, dog and nonhuman primates and (2) provides a review of our current understanding of the roles of gonadotropins and androgens. The response of the rodent testis to endocrine disturbances is clearly different from that of dog and primates with different germ cell types and spermatogenic stages being affected initially and also that the end-stage spermatogenic involution is more pronounced in dog and primates compared to rodents. Luteinizing hormone (LH)/testosterone and follicle-stimulating hormone (FSH) are the pivotal endocrine factors controlling testicular functions. The relative importance of either hormone is somewhat different between rodents and primates. Generally, however, both LH/testosterone and FSH are necessary for quantitatively normal spermatogenesis, at least in non-seasonal species. PMID:26413400

  8. Can the fall in serum FSH during coasting in IVF/ICSI predict clinical outcomes?

    PubMed

    Datta, Adrija Kumar; Zosmer, Ariel; Tozer, Amanda; Sabatini, Luca; Davis, Colin; Al-Shawaf, Talha

    2012-05-01

    This retrospective cohort study determined whether the total falls in serum FSH and oestradiol concentrations from start to end of coasting in IVF/intracytoplasmic sperm injection could predict clinical outcomes. Ninety-nine cycles, with gonadotrophin-releasing hormone-agonist down-regulation where coasting with serial serum oestradiol and FSH monitoring was adopted due to risk of severe ovarian hyperstimulation syndrome, were consecutively included. The primary clinical outcome was live-birth rate (LBR); other outcomes measured were number of oocytes retrieved and fertilization, implantation and clinical pregnancy rates. LBR for FSH fall>10 IU/l compared with 5-10 and<5 IU/l were 45.4% versus 22.0% and 25.0%, respectively. Mean serum FSH fall was similar with and without live birth (8.4 ± 6.2 versus 7.3 ± 5.0 IU/l) as were mean oestradiol and FSH concentrations on HCG administration, oestradiol fall, percentage fall in FSH/oestradiol and duration of coasting. None of the variables efficiently predicted live birth on regression analysis. The AUC of FSH fall was 0.53 at 11.0 IU/l. Basal FSH, starting and total gonadotrophin dose and duration of coasting were positively correlated with FSH fall. A potentially clinically important association between live birth and FSH fall during coasting was apparent, which requires further evaluation. The purpose of this retrospective cohort study was to determine whether the magnitude of fall in the serum FSH and oestradiol concentrations from start to end of coasting in IVF/intracytoplasmic sperm injection cycles could predict the clinical outcomes. Gonadotrophin-releasing hormone-agonist down-regulated cycles (n=99), where coasting with serial serum oestradiol and FSH monitoring was adopted due to risk of ovarian hyperstimulation, were consecutively included. Live birth was the primary clinical outcome measured; number of oocytes retrieved and fertilization, implantation and clinical pregnancy rates were the other outcomes

  9. FSH aggravates bone loss in ovariectomised rats with experimental periapical periodontitis

    PubMed Central

    Qian, Hua; Guan, Xiaoyue; Bian, Zhuan

    2016-01-01

    Periapical bone loss is one of the prominent pathological and clinical features of periapical periodontitis. Previous studies have demonstrated that follicle-stimulating hormone (FSH) could directly affect skeletal remodelling by stimulating the formation and the function of osteoclasts in vitro and in vivo. However, the effect of FSH on periapical bone loss remained to be fully elucidated. In the current study, a rat model was established in order to verify the effect of FSH in experimental periapical lesions. It was identified that FSH aggravated the bone loss of periapical lesions. In addition, RANKL-, TRAP-, TNF-α- and IL-1β-positive cells were increased significantly in FSH-treated groups, which indicated that the function of FSH in bone loss may be mediated through the increasing activity of osteoclasts and the increased secretion of inflammatory cytokines. The results of the current study suggested that FSH, independent of oestrogen, may aggravate periapical bone loss by FSH receptors, which may serve an important role in the immune and inflammatory response of the host to root canal and periradicular infection during menopause. PMID:27510616

  10. A study on superovulation using FSH and eCG in Awassi ewes.

    PubMed

    Azawi, Osama Ibrahim; Al-Mola, M K M A

    2010-06-01

    The present study was conducted to evaluate superovulatory treatments in Awassi ewes by eCG and FSH. High number of unovulated follicles (P < 0.05) was observed in ewes treated with eCG in non-breeding season. It could be concluded that using FSH to induce superovulation in Awassi ewes is better than eCG.

  11. [Synthèse of the LH-FSH-RH (author's transl)].

    PubMed

    Loffet, A; Durieux, J P

    1976-01-01

    The Luteinizing hormone-releasing hormone (LH-RH), and the follicle stimulating hormone-releasing hormone (FSH-RH) have been synthesized by a new procedure involving solid phase and conventional solution coupling. The release of LH and FSH by this synthetic product has been measured in both laboratory animals and in men. Results coincide exactly with those in the published literature.

  12. FSH protects mouse granulosa cells from oxidative damage by repressing mitophagy

    PubMed Central

    Shen, Ming; Jiang, Yi; Guan, Zhiqiang; Cao, Yan; Sun, Shao-chen; Liu, Honglin

    2016-01-01

    Oxidative stress has been implicated in triggering granulosa cell (GC) death during follicular atresia. Recent studies suggested that follicle-stimulating hormone (FSH) has a pivotal role in protecting GCs from oxidative injury, although the exact mechanism remains largely unknown. Here, we report that FSH promotes GC survival by inhibiting oxidative stress-induced mitophagy. The loss of GC viability caused by oxidative stress was significantly reduced after FSH treatment, which was correlated with impaired activation of mitophagy upon oxidative stress. Compared with FSH treatment, blocking mitophagy displayed approximate preventive effect on oxidative stress-induced GC death, but FSH did not further restore viability of cells pretreated with mitophagy inhibitor. Importantly, FSH suppressed the induction of serine/threonine kinase PINK1 during oxidative stress. This inhibited the mitochondrial translocation of the E3 ligase Parkin, which is required for the subsequent clearance of mitochondria, and ultimately cell death via mitophagy. In addition, knocking down PINK1 using RNAi confirmed the role of the FSH-PINK1-Parkin-mitophagy pathway in regulating GC survival under oxidative conditions. These findings introduce a novel physiological function of FSH in protecting GCs against oxidative damage by targeting PINK1-Parkin-mediated mitophagy. PMID:27901103

  13. [Variations in the molecular forms of LH and FSH during the normal ovarian cycle].

    PubMed

    Hernandez-Valencia, M; Mason, M; Eugenia Fonseca, M; Zarate, A

    1996-03-01

    FSH and LH concentrations during the menstrual cycle are well known, showing characteristically a midcycle surge in LH as well as in less degree in FSH. At the present there is an increasing interest in studying variations of the moleculoar forms of both gonadotropins. We have studied the chromatographic profile of FSH and LH in sera obtained from women regularly ovulating and in patients with anovulatory cycles by the use of gel column chromatography. It was observed the predominance of LH 32-34 kDa at midcycle and the heterogeneous FSH profile during the ovarian cycle. In sera from anovulatory patients the chromatographic profile was even more irregular for both LH and FSH. It is concluded that LH 32-34 kDa may be the more biological isoform in turn related with the process of ovulation.

  14. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    PubMed

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

  15. Renal cell carcinoma metastatic to a pituitary FSH/LH adenoma: case report and review of the literature.

    PubMed

    Magnoli, Francesca; Finzi, Giovanna; Riva, Cristina; Capella, Carlo

    2014-12-01

    Abstract Metastases to the pituitary occur more frequently in patients with widespread cancer and mainly involve the posterior lobe. A few cases of metastatic carcinoma to a pituitary adenoma have been described so far. Here, the authors present an additional case of a clear cell renal cell carcinoma (CCRCC) metastatic to a FSH/LH/α-subunit pituitary adenoma and systematically review the literature. Immunohistochemistry and electron microscopy were performed to characterize both neoplastic components at the morphological level. Moreover, it was hypothesized that expression of VEGF and of the corresponding receptor VEGFR1 could be implicated in the development of the carcinomatous metastasis within the adenoma.

  16. A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles.

    PubMed

    Allegra, Adolfo; Marino, Angelo; Volpes, Aldo; Coffaro, Francesco; Scaglione, Piero; Gullo, Salvatore; La Marca, Antonio

    2017-01-23

    The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8-14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

  17. Gene Expression Patterns in Human Liver Cancers

    PubMed Central

    Chen, Xin; Cheung, Siu Tim; So, Samuel; Fan, Sheung Tat; Barry, Christopher; Higgins, John; Lai, Kin-Man; Ji, Jiafu; Dudoit, Sandrine; Ng, Irene O.L.; van de Rijn, Matt; Botstein, David; Brown, Patrick O.

    2002-01-01

    Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Using cDNA microarrays to characterize patterns of gene expression in HCC, we found consistent differences between the expression patterns in HCC compared with those seen in nontumor liver tissues. The expression patterns in HCC were also readily distinguished from those associated with tumors metastatic to liver. The global gene expression patterns intrinsic to each tumor were sufficiently distinctive that multiple tumor nodules from the same patient could usually be recognized and distinguished from all the others in the large sample set on the basis of their gene expression patterns alone. The distinctive gene expression patterns are characteristic of the tumors and not the patient; the expression programs seen in clonally independent tumor nodules in the same patient were no more similar than those in tumors from different patients. Moreover, clonally related tumor masses that showed distinct expression profiles were also distinguished by genotypic differences. Some features of the gene expression patterns were associated with specific phenotypic and genotypic characteristics of the tumors, including growth rate, vascular invasion, and p53 overexpression. PMID:12058060

  18. Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH.

    PubMed

    Karakaya, Cengiz; Guzeloglu-Kayisli, Ozlem; Hobbs, Rebecca J; Gerasimova, Tsilya; Uyar, Asli; Erdem, Mehmet; Oktem, Mesut; Erdem, Ahmet; Gumuslu, Seyhan; Ercan, Deniz; Sakkas, Denny; Comizzoli, Pierre; Seli, Emre; Lalioti, Maria D

    2014-07-01

    Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

  19. Chorionic gonadotropin and its receptor are both expressed in human retina, possible implications in normal and pathological conditions.

    PubMed

    Dukic-Stefanovic, Sladjana; Walther, Jan; Wosch, Sebastian; Zimmermann, Gerolf; Wiedemann, Peter; Alexander, Henry; Claudepierre, Thomas

    2012-01-01

    Extra-gonadal role of gonadotropins has been re-evaluated over the last 20 years. In addition to pituitary secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH), the CNS has been clearly identified as a source of hCG acting locally to influence behaviour. Here we demonstrated that human retina is producing this gonadotropin that acts as a neuroactive molecule. Müller glial and retinal pigmented epithelial (RPE) cells are producing hCG that may affects neighbour cells expressing its receptor, namely cone photoreceptors. It was previously described that amacrine and retinal ganglion (RGC) cells are targets of the gonadotropin releasing hormone that control the secretion of all gonadotropins. Therefore our findings suggest that a complex neuroendocrine circuit exists in the retina, involving hCG secreting cells (glial and RPE), hCG targets (photoreceptors) and hCG-release controlling cells (amacrine and RGC). The exact physiological functions of this circuit have still to be identified, but the proliferation of photoreceptor-derived tumor induced by hCG demonstrated the need to control this neuroendocrine loop.

  20. [Hospital humanization as an expression of ethics].

    PubMed

    Backes, Dirce Stein; Lunardi, Valéria Lerch; Lunardi, Wilson D Filho

    2006-01-01

    The practice of health professionals in the hospital environment has been loosing its human characteristics in view of the care delivered to the disease rather than to the sick being, and also in view of the increasing technological complexity, associated with increasing costs. Ethics requires the implementation of a reflective process concerning the principles, values, rights, and duties ruling the practice of health professionals, the latter including the dimension of care from a humanized perspective. Thus, this study aimed to reflect on ethical considerations upon which humanization actions must be grounded, highlighting the importance of a human dimension in professional relations.

  1. [Human angiogenin: expression, purification, biological assay].

    PubMed

    Yang, H; Zhang, Y Q; Yan, Z; Han, W; Yao, L B; Su, C Z

    2001-01-01

    Angiogenin cDNA was obtained by RT-PCR, and cloned into the fusion expression vector pRSETB. The recombinant Angiogenin protein was fused with His6 at its N-terminal and expressed as inclusion body. The expression level was about 10% of the total bacteria protein. After dissolved in 8 mol/L urea, the recombinant protein was purified by Ni2(+)-NTA chelating resin, according to the high affinity of His6 with Ni2+. The biological assay indicated that purified rhANG could induced the new blood vessel formation of CAM and degraded tRNA in vitro.

  2. Fasting lowers gastrin-releasing peptide and FSH mRNA in the ovine anterior pituitary gland

    USDA-ARS?s Scientific Manuscript database

    Estrogen receptor beta (ER-ß), LH, and FSH are important mediators of reproduction. FSH stimulates follicle recruitment and development. During anorexia, serum concentrations of FSH and LH decrease. Gastrin-releasing peptide (GRP), neuromedin B (NMB), peroxisome proliferator-activated receptor-gamma...

  3. Fasting lowers gastrin-releasing peptide and Fsh mRNA in the ovine anterior pituitary gland

    USDA-ARS?s Scientific Manuscript database

    Estrogen receptor beta (ER-ß), LH, and FSH are important mediators of reproduction. FSH stimulates follicle recruitment and development. During anorexia, serum concentrations of FSH and LH decrease. Gastrin-releasing peptide (GRP), neuromedin B (NMB), peroxisome proliferator-activated receptor-gamma...

  4. Human Facial Expressions as Adaptations:Evolutionary Questions in Facial Expression Research

    PubMed Central

    SCHMIDT, KAREN L.; COHN, JEFFREY F.

    2007-01-01

    The importance of the face in social interaction and social intelligence is widely recognized in anthropology. Yet the adaptive functions of human facial expression remain largely unknown. An evolutionary model of human facial expression as behavioral adaptation can be constructed, given the current knowledge of the phenotypic variation, ecological contexts, and fitness consequences of facial behavior. Studies of facial expression are available, but results are not typically framed in an evolutionary perspective. This review identifies the relevant physical phenomena of facial expression and integrates the study of this behavior with the anthropological study of communication and sociality in general. Anthropological issues with relevance to the evolutionary study of facial expression include: facial expressions as coordinated, stereotyped behavioral phenotypes, the unique contexts and functions of different facial expressions, the relationship of facial expression to speech, the value of facial expressions as signals, and the relationship of facial expression to social intelligence in humans and in nonhuman primates. Human smiling is used as an example of adaptation, and testable hypotheses concerning the human smile, as well as other expressions, are proposed. PMID:11786989

  5. Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.

    PubMed

    Silva, J M; Hamel, M; Sahmi, M; Price, C A

    2006-12-01

    The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.

  6. Genes Expressed in Human Tumor Endothelium

    NASA Astrophysics Data System (ADS)

    St. Croix, Brad; Rago, Carlo; Velculescu, Victor; Traverso, Giovanni; Romans, Katharine E.; Montgomery, Elizabeth; Lal, Anita; Riggins, Gregory J.; Lengauer, Christoph; Vogelstein, Bert; Kinzler, Kenneth W.

    2000-08-01

    To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.

  7. Trehalose expression confers desiccation tolerance on human cells.

    PubMed

    Guo, N; Puhlev, I; Brown, D R; Mansbridge, J; Levine, F

    2000-02-01

    Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

  8. Bone loss in ovariectomized rats: dominant role for estrogen but apparently not for FSH.

    PubMed

    Rouach, V; Katzburg, S; Koch, Y; Stern, N; Somjen, D

    2011-01-01

    Estrogen deficiency as the sole factor underlying post-menopausal osteoporosis was challenged, in light of reports that both follicular stimulation hormone (FSH) receptor and FSHβ knockout mice were resistant to bone loss, suggesting a detrimental role for FSH. We assessed whether lowering FSH levels by gonadotropin realizing (GnRH) analog decapeptyl in ovariectomized female rats (OVX) affects bone. Wistar-derived 25 days old OVX female rats were injected for 10 weeks with estradiol-17β (E(2)), with GnRH analog (decapeptyl) or with both. FSH and luteinizing hormone (LH) serum levels were markedly increased in OVX rats, with smaller growth plates with disrupted architecture; heavy infiltration of bone marrow with numerous adipocytes and reduced thickness of cortical bone. In OVX rats treated with E(2), FSH, and LH levels were intermediate, the tibia was similar to that of intact rats, but there was reduced thickness of cortical bone. In decapeptyl treated OVX rats, FSH and LH levels were suppressed, the organization of growth plate and the trabecular bone were disrupted, and there were fewer proliferative and chondroblastic cells and a large adipocytes population in bone marrow, but an increased trabecular bone volume (TBV). In the E(2) + decapeptyl treatment, FSH and LH levels were suppressed, with partially restored growth plate architecture and improved TBV. In conclusion, E(2) deficiency is the dominant factor impairing bone loss in OVX and concomitant changes in FSH/LH levels achieved by decapeptyl have some modulating, though complex role in this setting. The role of high FSH levels in post-menopausal bone loss requires further investigation using combined sub-optimal doses of the different hormones.

  9. An Investigation on a Novel Anti-tumor Fusion Peptide of FSH33-53-IIKK

    PubMed Central

    Yang, Runlin; Liu, Ping; Pan, Donghui; zhang, Pengjun; Bai, Zhicheng; Xu, Yuping; Wang, Lizhen; Yan, Junjie; Yan, Yongjun; Liu, Xingdang; Yang, Min

    2016-01-01

    A novel fusion peptide FSH33-53-IIKK was designed and expected to combine the follicle stimulating hormone receptor (FSHR) targeting and tumor toxicity. In vitro and in vivo study showed the anti-tumor activity of FSH33-53-IIKK was enhanced compared to that of IIKK only. FSH33-53-IIKK could inhibit the growth of tumor via apoptosis and autophagy pathways. In summary, combining the tumor marker-target peptide and anti-tumor peptide together may be an efficient way to search for better anti-tumor candidates. PMID:27313792

  10. Evolution and proximate expression of human paternal investment.

    PubMed

    Geary, D C

    2000-01-01

    In more than 95% of mammalian species, males provide little direct investment in the well-being of their offspring. Humans are one notable exception to this pattern and, to date, the factors that contributed to the evolution and the proximate expression of human paternal care are unexplained (T. H. Clutton-Brock, 1989). The nature, extent, and influence of human paternal investment on the physical and social well-being of children are reviewed in light of the social and ecological factors that are associated with paternal investment in other species. On the basis of this review, discussion of the evolution and proximate expression of human paternal investment is provided.

  11. Mice Expressing RHAG and RHD Human Blood Group Genes

    PubMed Central

    Goossens, Dominique; da Silva, Nelly; Metral, Sylvain; Cortes, Ulrich; Callebaut, Isabelle; Picot, Julien; Mouro-Chanteloup, Isabelle; Cartron, Jean-Pierre

    2013-01-01

    Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RhAG and RhD erythrocyte membrane proteins in the presence and, for human RhAG, in the absence, of mouse Rhag. Human RhAG associates with mouse Rh but not mouse Rhag on red blood cells. In Rhag knockout mice transgenic for human RHAG, the mouse Rh protein is “rescued” (re-expressed), and co-immunoprecipitates with human RhAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of β-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RhAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-Rhag knockout mice we have developed previously. PMID:24260394

  12. Novel Action of FSH on Stem Cells in Adult Mammalian Ovary Induces Postnatal Oogenesis and Primordial Follicle Assembly

    PubMed Central

    Parte, Seema; Patel, Hiren; Sriraman, Kalpana; Zaveri, Kusum; Hinduja, Indira

    2016-01-01

    Adult mammalian ovary has been under the scanner for more than a decade now since it was proposed to harbor stem cells that undergo postnatal oogenesis during reproductive period like spermatogenesis in testis. Stem cells are located in the ovary surface epithelium and exist in adult and menopausal ovary as well as in ovary with premature failure. Stem cells comprise two distinct populations including spherical, very small embryonic-like stem cells (VSELs which express nuclear OCT-4 and other pluripotent and primordial germ cells specific markers) and slightly bigger ovarian germ stem cells (OGSCs with cytoplasmic OCT-4 which are equivalent to spermatogonial stem cells in the testes). These stem cells have the ability to spontaneously differentiate into oocyte-like structures in vitro and on exposure to a younger healthy niche. Bone marrow may be an alternative source of these stem cells. The stem cells express FSHR and respond to FSH by undergoing self-renewal, clonal expansion, and initiating neo-oogenesis and primordial follicle assembly. VSELs are relatively quiescent and were recently reported to survive chemotherapy and initiate oogenesis in mice when exposed to FSH. This emerging understanding and further research in the field will help evolving novel strategies to manage ovarian pathologies and also towards oncofertility. PMID:26635884

  13. [Antimicrobial resistance of coliform isolates from expressed human milk].

    PubMed

    Novak, F R; Almeida, J A; Asensi, M D; Moraes, B A; dos Prazeres Rodrigues, D

    2001-01-01

    The dispersion of potentially pathogenic, antibiotic-resistant microorganisms via expressed human milk can be considered a risk factor. The aim of this study was to contribute to a better understanding of coliform isolates from expressed human milk and their antimicrobial resistance profiles. The sampling scheme followed a totally randomized design, using 837 samples of expressed human milk. Of these, 71 (8.48%) were identified as contaminated with total coliforms, although in none of the samples did the population exceed 1.0x10(3) MPN/ml. Most of the microorganisms isolated (91.6%) belonged to only two species, Enterobacter cloacae and Klebsiella pneumoniae, which when subjected to antibiograms, revealed that several strains showed prior resistance to some of the antimicrobials tested. Coliforms may grow in expressed human milk if it is improperly stored, depleting protection factors and reducing the milk's nutritional value.

  14. Expression of the alpha subunit of human chorionic gonadotropin is specifically correlated with tumorigenic expression in human cell hybrids.

    PubMed Central

    Stanbridge, E J; Rosen, S W; Sussman, H H

    1982-01-01

    The expression of HeLa parent phenotype protein markers, the alpha subunit of human chorionic gonadotropin and placental alkaline phosphatase isoenzymes, has been evaluated in paired tumorigenic and nontumorigenic HeLa-fibroblast human cell hybrids. Both of these proteins have been used clinically as markers of malignancy. The results showed that both are expressed in the hybrids. Expression of the gonadotropin subunit in the hybrids is specifically correlated with tumorigenicity; the placental alkaline phosphatase isoenzyme showed no such correlation and was expressed in both tumorigenic and nontumorigenic hybrids. PMID:6959112

  15. Hemoglobin enhances tissue factor expression on human malignant cells.

    PubMed

    Siddiqui, F A; Amirkhosravi, A; Amaya, M; Meyer, T; Biggerstaff, J; Desai, H; Francis, J L

    2001-04-01

    Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

  16. Human basophils express CD22 without expression of CD19.

    PubMed

    Han, K; Kim, Y; Lee, J; Lim, J; Lee, K Y; Kang, C S; Kim, W I; Kim, B K; Shim, S I; Kim, S M

    1999-11-01

    Even modern automatic cell counters cannot count basophils precisely. Therefore, we need a rapid, accurate, precise, and easy method for counting basophils. Using flow cytometry, basophils (CD22+/CD19-) and B cells (CD22+/CD19+) were counted. Within a large lymphocyte light scatter gate, % basophils (G%baso) and % B cells (G%B) were determined from the total count. Another method of analysis was to make two regions (R1 for basophils and R2 for B cells) and to determine in those the % basophils (R1%baso) and % B cells (R2%B) without gating. The flow cytometric basophil counts of the blood of 21 normal controls and 43 chronic myelogenous leukemia (CML) patients were compared with manual basophil count (Ma%baso) and basophil count by Coulter electronic cell counter (Hialeah, FL) (Auto%baso). CD22+/CD19- cells were sorted by a FACSCalibur (Becton Dickinson, San Jose, CA). The G%baso of all samples was 4.66 +/- 5.35%, and R1%baso was 4.23 +/- 4.88%, and they were well-correlated (r = 0.996, P < 0.001). The G%B of all samples was 1.55 +/- 1.68%, and R2%B was 1.59 +/- 1.67%, and they were also well-correlated (r = 0.993, P < 0.001). Their correlation was better in normal controls than in CML. G%baso was well-correlated to Ma%baso (r = 0.827) and Auto%baso (r = 0.806), and R1%baso was well-correlated to Ma%baso (r = 0.831) but showed poor correlation to Auto%baso (r = 0.734). Auto%baso revealed the poorest correlation to Ma%baso (r = 0.692). The sorted CD22+/CD19- cells were all basophils (99.48 +/- 0.30%), and they revealed CD13, CD33, and dim CD45 expression, whereas CD3, CD14, CD16, and HLA-DR were not detected on them. We discovered a specific marker combination to identify basophils (CD22+/CD19-), and we suggest that flow cytometric analysis using these markers is an easy, reliable, and accurate method of basophil counting. Copyright 1999 Wiley-Liss, Inc.

  17. Epigenetic Control of Retrotransposon Expression in Human Embryonic Stem Cells▿

    PubMed Central

    Macia, Angela; Muñoz-Lopez, Martin; Cortes, Jose Luis; Hastings, Robert K.; Morell, Santiago; Lucena-Aguilar, Gema; Marchal, Juan Antonio; Badge, Richard M.; Garcia-Perez, Jose Luis

    2011-01-01

    Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated. PMID:21041477

  18. Immunohistochemical characterization of FHIT expression in normal human tissues

    PubMed Central

    Kujan, Omar; Abuderman, Abdulwahab; Al-Shawaf, Ahmad Zahi

    2016-01-01

    Background Fragile histidine triad (FHIT) is a tumor suppressor gene that is commonly inactivated in human tumors. Interestingly, the normal pattern of FHIT expression is largely unknown. Aim This study is aimed to characterize the expression of FHIT protein in normal human tissues. Materials and methods A total of 119 normal human tissue specimens were analyzed for the FHIT expression using immunohistochemistry technique. The inclusion criteria included: normal/inflammatory tissue with no evidence of cellular atypia. Results All studied specimens were stained positively with FHIT and showed either nuclear or cytoplasmic expression. Interestingly, the pattern of FHIT staining was similar among different specimens from each organ. FHIT is located predominantly in the nucleus, although cytoplasmic staining is also present in some cell types. Oral squamous epithelium, breast ductal epithelium, squamous and tubal metaplastic epithelium of the uterine cervix, esophageal squamous epithelium, salivary glands, and bronchial epithelia all strongly expressed the nuclear protein. In connective tissue, FHIT has shown strong cytoplasmic expression in histocytes including macrophages and dendritic cells, fibroblasts, and myofibroblasts. Conclusion Documentation of the pattern of FHIT expression in normal tissues will contribute to our understanding of the normal function of this protein and to interpretation of potentially altered FHIT expression in human tumors. PMID:28250975

  19. Epigenetic control of retrotransposon expression in human embryonic stem cells.

    PubMed

    Macia, Angela; Muñoz-Lopez, Martin; Cortes, Jose Luis; Hastings, Robert K; Morell, Santiago; Lucena-Aguilar, Gema; Marchal, Juan Antonio; Badge, Richard M; Garcia-Perez, Jose Luis

    2011-01-01

    Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.

  20. Developmental regulation of CFTR expression during human nephrogenesis.

    PubMed

    Devuyst, O; Burrow, C R; Schwiebert, E M; Guggino, W B; Wilson, P D

    1996-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein are expressed in proximal and distal tubules of the human kidney, but CFTR expression pattern during human nephrogenesis is unknown. We have now studied CFTR expression in fetal kidneys by immunohistochemistry and Western blot analysis, using six antibodies against human CFTR. CFTR was expressed in 12-wk human fetal kidneys, mostly in the apical membrane region of the ureteric bud epithelial cells. By 15 wk, CFTR was also diffusely expressed throughout the cytoplasm of proximal tubules and loops of Henle. No glomerular staining was seen at any state. From 15 to 24 wk of gestation this staining pattern remained constant and also included immunoreactivity of the transitional epithelium. Western blot for CFTR was performed on membrane extracts of human fetal kidneys, using T84 cells as a positive control. A 165-kDa protein corresponding to the predicted size of CFTR was seen at 13 wk and throughout development. We also observed a 75-kDa protein that was distinctly regulated during development. This protein was detected with several antibodies against the first half of CFTR (including the regulatory "R" domain) but not with a COOH-terminal-specific antibody and had the predicted size of a functional splice variant of CFTR identified in the human kidney. These results show the complex regulation of CFTR during nephrogenesis and raise the question of the respective roles of the full-length and the splice variant CFTR proteins in the human kidney.

  1. Regulated expression of the human gastrin gene in mice.

    PubMed

    Mensah-Osman, Edith; Labut, Ed; Zavros, Yana; El-Zaatari, Mohamad; Law, David J; Merchant, Juanita L

    2008-11-29

    Gastrin is secreted from neuroendocrine cells residing in the adult antrum called G cells, but constitutively low levels are also expressed in the duodenum and fetal pancreas. Gastrin normally regulates gastric acid secretion by stimulating the proliferation of enterochromaffin-like cells and the release of histamine. Gastrin and progastrin forms are expressed in a number of pathological conditions and malignancies. However, the DNA regulatory elements in the human versus the mouse gastrin promoters differ suggesting differences in their transcriptional control. Thus, we describe here the expression of the human gastrin gene using a bacterial artificial chromosome (BAC) in the antral and duodenal cells of gastrin null mice. All 5 founder lines expressed the 253 kb human gastrin BAC. hGasBAC transgenic mice were bred onto a gastrin null background so that the levels of human gastrin peptide could be analyzed by immunohistochemistry and radioimmunoassay without detecting endogenous mouse gastrin. We have shown previously that chronically elevated gastrin levels suppress somatostatin. Indeed, infusion of amidated rat gastrin depressed somatostatin levels, stimulated gastric acid secretion and an increase in the numbers of G cells in the antrum and duodenum. In conclusion, human gastrin was expressed in mouse enteroendocrine cells and was regulated by somatostatin. This mouse model provides a unique opportunity to study regulation of the human gastrin promoter in vivo by somatostatin and possibly other extracellular regulators contributing to our understanding of the mechanisms involved in transcriptional control of the human gene.

  2. Gene profile identifies zinc transporters differentially expressed in normal human organs and human pancreatic cancer.

    PubMed

    Yang, J; Zhang, Y; Cui, X; Yao, W; Yu, X; Cen, P; Hodges, S E; Fisher, W E; Brunicardi, F C; Chen, C; Yao, Q; Li, M

    2013-03-01

    Deregulated expression of zinc transporters was linked to several cancers. However, the detailed expression profile of all human zinc transporters in normal human organs and in human cancer, especially in pancreatic cancer is not available. The objectives of this study are to investigate the complete expression patterns of 14 ZIP and 10 ZnT transporters in a large number of normal human organs and in human pancreatic cancer tissues and cell lines. We examined the expression patterns of ZIP and ZnT transporters in 22 different human organs and tissues, 11 pairs of clinical human pancreatic cancer specimens and surrounding normal/benign tissues, as well as 10 established human pancreatic cancer cell lines plus normal human pancreatic ductal epithelium (HPDE) cells, using real time RT-PCR and immunohistochemistry. The results indicate that human zinc transporters have tissue specific expression patterns, and may play different roles in different organs or tissues. Almost all the ZIPs except for ZIP4, and most ZnTs were down-regulated in human pancreatic cancer tissues compared to the surrounding benign tissues. The expression patterns of individual ZIPs and ZnTs are similar among different pancreatic cancer lines. Those results and our previous studies suggest that ZIP4 is the only zinc transporter that is significantly up-regulated in human pancreatic cancer and might be the major zinc transporter that plays an important role in pancreatic cancer growth. ZIP4 might serve as a novel molecular target for pancreatic cancer diagnosis and therapy.

  3. Effects of neonatal androgenization on the chromatofocusing pattern of anterior pituitary FSH in the female rat.

    PubMed

    Ulloa-Aguirre, A; Damián-Matsumura, P; Espinoza, R; Dominguez, R; Morales, L; Flores, A

    1990-08-01

    Anterior pituitary glands were removed from neonatally androgenized (100 micrograms testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7.5-4.0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7.5-4.0, as well as an additional FSH form obtained after the addition of 1.0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5-30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5.9-5.0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39.2 +/- 0.6%; androgenized, 23.1 +/- 0.9% of total immunoactivity recovered; P less than 0.03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P less than 0.01). In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23.2-28.8% of total) focused within a pH range of 5.9-5.0, whilst only 10.4-11.6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P less than 0.05). In control animals decapitated during the morning of pro-oestrus and oestrus, 10-26% of FSH focused within the most alkaline region (pI 7.5-6.0); the chromatofocusing

  4. Mucin gene expression in human middle ear epithelium.

    PubMed

    Kerschner, Joseph Edward

    2007-09-01

    To investigate the expression of recently identified human mucin genes in human middle ear epithelial (MEE) specimens from in vivo middle ear (ME) tissue and to compare this mucin gene expression with mucin gene expression in an immortalized cell culture in vitro source of human MEE. Human MEE was harvested as in vivo specimens, and human MEE cell cultures were established for in vitro experimentation. RNA was extracted from MEE and primers designed for reverse-transcription polymerase chain reaction to assess for mucin gene MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 expression. Mucin gene expression in the in vivo and in vitro ME tissue was compared against tissues with known expression of the mucin genes in question. Mucin genes MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC9, MUC11, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 were identified and expressed in both the in vivo and in vitro samples of MEE. Mucin genes MUC6, MUC12, and MUC17 were not identified in either tissue samples. Many of the mucin genes that have been recently identified are expressed in human MEE. These genes are expressed in a similar manner in both in vivo and in vitro models. Understanding the mechanisms in which these genes regulate the physiology and pathophysiology of MEE will provide a more thorough understanding of the molecular mechanics of the MEE and disease conditions such as otitis media.

  5. Expressive Writing: Enhancing the Emotional Intelligence of Human Services Majors

    ERIC Educational Resources Information Center

    Castillo, Yuleinys; Fischer, Jerome M.

    2017-01-01

    The skills and tasks in the human services field are highly connected to emotional intelligence abilities. The purpose of the study was to investigate the effect of an expressive writing program involving human service students in an undergraduate rehabilitation services course. The program was developed to enhance their emotional intelligence.…

  6. Expression of human antibodies in eukaryotic micro-algae.

    PubMed

    Mayfield, Stephen P; Franklin, Scott E

    2005-03-07

    Protein based therapeutics have enjoyed great success over the past decade. Unfortunately, with this clinical success comes a heavy price tag, owing to the inherently high costs of capitalization and production using mammalian cell fermentation. To address this problem, we have begun developing a system for the expression of recombinant proteins in the unicellular eukaryotic green algae, Chlamydomonas reinhardtii, leading to the production of human IgA single chain antibodies. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian based expression systems for several reasons, including an ability to rapidly obtain stable plastid and nuclear transformants, coupled with inherently low costs of capitalization and production.

  7. Lateralization for dynamic facial expressions in human superior temporal sulcus.

    PubMed

    De Winter, François-Laurent; Zhu, Qi; Van den Stock, Jan; Nelissen, Koen; Peeters, Ronald; de Gelder, Beatrice; Vanduffel, Wim; Vandenbulcke, Mathieu

    2015-02-01

    Most face processing studies in humans show stronger activation in the right compared to the left hemisphere. Evidence is largely based on studies with static stimuli focusing on the fusiform face area (FFA). Hence, the pattern of lateralization for dynamic faces is less clear. Furthermore, it is unclear whether this property is common to human and non-human primates due to predisposing processing strategies in the right hemisphere or that alternatively left sided specialization for language in humans could be the driving force behind this phenomenon. We aimed to address both issues by studying lateralization for dynamic facial expressions in monkeys and humans. Therefore, we conducted an event-related fMRI experiment in three macaques and twenty right handed humans. We presented human and monkey dynamic facial expressions (chewing and fear) as well as scrambled versions to both species. We studied lateralization in independently defined face-responsive and face-selective regions by calculating a weighted lateralization index (LIwm) using a bootstrapping method. In order to examine if lateralization in humans is related to language, we performed a separate fMRI experiment in ten human volunteers including a 'speech' expression (one syllable non-word) and its scrambled version. Both within face-responsive and selective regions, we found consistent lateralization for dynamic faces (chewing and fear) versus scrambled versions in the right human posterior superior temporal sulcus (pSTS), but not in FFA nor in ventral temporal cortex. Conversely, in monkeys no consistent pattern of lateralization for dynamic facial expressions was observed. Finally, LIwms based on the contrast between different types of dynamic facial expressions (relative to scrambled versions) revealed left-sided lateralization in human pSTS for speech-related expressions compared to chewing and emotional expressions. To conclude, we found consistent laterality effects in human posterior STS but not

  8. Hypersecretion of FSH in infant boys and girls born small for gestational age.

    PubMed

    Ibáñez, Lourdes; Valls, Carme; Cols, Maria; Ferrer, Angela; Marcos, Maria Victoria; De Zegher, Francis

    2002-05-01

    Prenatal growth restraint, as reflected in a low birthweight for gestational age, is a risk factor for postpubertal FSH hypersecretion and for reduced gonadal size. The ontogeny of the low-birthweight effect on the FSH-inhibin B feedback loop is unknown. Infancy is an episode of choice to study the possibility of an early low-birthweight effect on the FSH-inhibin B loop because this phase is characterized by high activity within the gonadal axis. We assessed serum concentrations of FSH and inhibin B in 46 infants [26 girls and 20 boys; mean age, 4 months; range, 3-6 months; 17 appropriate for gestational age (AGA), 29 small for gestational age (SGA); mean birthweight, 3.2 kg for AGA vs. 2.3 kg for SGA], together with circulating levels of LH, E2, and free androgen index. In SGA girls and boys, serum FSH levels were 2- and 4-fold higher (P < 0.001), respectively, than in AGA controls of the same gender (7.3 +/- 0.9 vs. 3.8 +/- 0.4 IU/ml and 2.9 +/- 0.5 vs. 0.7 +/- 0.2 IU/ml). Serum LH, inhibin B, and free androgen index/E2 concentrations were similar in AGA and SGA infants. In conclusion, prenatal growth restraint was found to be followed by elevated serum FSH concentrations in infant girls and boys. SGA infants seem to need an augmented FSH drive to fulfill inhibin B requirements on the afferent side of the feedback loop. The late-endocrine correlates of early growth restraint are herewith extended to include the main axis of reproduction in both genders. It remains to be studied whether FSH hypersecretion in infancy is a marker of subsequent subfertility.

  9. FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence

    PubMed Central

    Franciosi, Federica; Manandhar, Shila

    2016-01-01

    A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3′ untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Ptenfl/fl:Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality. PMID:26653334

  10. Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

    SciTech Connect

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Mali, P.; Huhtaniemi, I.; Parvinen, M. )

    1990-05-01

    Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.

  11. FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence.

    PubMed

    Franciosi, Federica; Manandhar, Shila; Conti, Marco

    2016-02-01

    A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality.

  12. Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules

    SciTech Connect

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Perheentupa, A.; Huhtaniemi, I.; Parvinen, M. )

    1990-07-01

    Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.

  13. [Embryonic quality in the use of urofolitropin vs recombinant FSH for in vitro fertilization].

    PubMed

    Contreras Bretherton, C; David Vargas, R; Cedillo, F J; Vilchez, R; Das Neves, D; Bernal, M A; Verez Ruíz, J R; Stern Colin y Nunes, J J; Gutiérrez Nájar, A

    1999-05-01

    There were no differences in both groups as tho the age of the patients; received doses of both types of FSH, nor HMG; but there was as to the amount of captured ovocytes, amount, and quality, embrionary, in special 1+ 2+ in favor of the group that received urofolitropine, specially under 35 years of age. In this study there was better qualy and amount, embrionary, obtained with the use of urofolitropine, as compared with FSH recombinant for in vitro fertilization.

  14. Selective changes of retroelement expression in human prostate cancer.

    PubMed

    Goering, Wolfgang; Ribarska, Teodora; Schulz, Wolfgang A

    2011-10-01

    Retroelements constitute a large part of the human genome. These sequences are mostly silenced in normal cells, but genome-wide DNA hypomethylation in cancers might lead to their re-expression. Whether this re-expression really occurs in human cancers is largely unkown. We therefore investigated expression and DNA methylation of several classes of retroelements in human prostate cancer tissues and cell lines by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. The most striking finding was strong and generalized increased expression of the HERV-K_22q11.23 provirus in cancers, including de novo expression of a spliced accessory Np9 transcript in some tumors. In parallel, DNA methylation in the long terminal repeat (LTR) decreased. Conversely, HERVK17 expression was significantly diminished in cancer tissues, but this decrease was unrelated to LTR methylation. Expression of both proviruses was restricted to androgen-responsive prostate cancer cell lines and LTRs sequences containing steroid hormone-responsive elements bound the androgen receptor and conferred androgen responsiveness to reporter constructs. Expression of LINE-1 5'-untranslated region (UTR) and 3'-UTR sequences in prostate cancers rather decreased, despite significant hypomethylation of the internal LINE-1 promoter. Increased expression of the young AluYa5 and AluYb8 families was restricted to individual tumors. Our findings demonstrate a surprising specificity of changes in expression and DNA methylation of retroelements in prostate cancer. In particular, LINE-1 hypomethylation does not lead to generalized overexpression, but specific human endogenous retrovirus-K proviruses display conspicuous changes in their expression hinting at significant functions during prostate carcinogenesis.

  15. Decorin gene expression and its regulation in human keratinocytes

    SciTech Connect

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico; Kuri-Harcuch, Walid

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  16. Expression and distribution of endocan in human tissues.

    PubMed

    Zhang, S M; Zuo, L; Zhou, Q; Gui, S Y; Shi, R; Wu, Q; Wei, W; Wang, Y

    2012-04-01

    Endocan is a novel human endothelial cell specific molecule. Its expression is regulated by cytokines and vascular endothelial growth factor (VEGF). The distribution of endocan in normal human tissues, however, remains unclear. We examined the expression of endocan in normal human tissue using immunohistochemical stains. Endocan was expressed in actively proliferative or neogeneic tissues and cells such as glandular tissues, endothelium of neovasculature, bronchial epithelium, germinal centers of lymph nodes etc. Endocan was not present in silent or resting tissues or cells such as endothelium of great arteries and spleen etc. Our findings suggest that endocan may act as a marker for angiogenesis or oncogenesis and could be regarded as a candidate gene for inflammatory tissue, neoplasia, tumor development and metastasis. The expression level of endocan may assist early diagnosis and prognosis of some tumors.

  17. Differential gene expression in anatomical compartments of the human eye

    PubMed Central

    Diehn, Jennifer J; Diehn, Maximilian; Marmor, Michael F; Brown, Patrick O

    2005-01-01

    Background The human eye is composed of multiple compartments, diverse in form, function, and embryologic origin, that work in concert to provide us with our sense of sight. We set out to systematically characterize the global gene expression patterns that specify the distinctive characteristics of the various eye compartments. Results We used DNA microarrays representing approximately 30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. The distinctive patterns of expression in each compartment could be interpreted in relation to the physiology and cellular composition of each tissue. Notably, the sets of genes selectively expressed in the retina and in the lens were particularly large and diverse. Genes with roles in immune defense, particularly complement components, were expressed at especially high levels in the anterior segment tissues. We also found consistent differences between the gene expression patterns of the macula and peripheral retina, paralleling the differences in cell layer densities between these regions. Based on the hypothesis that genes responsible for diseases that affect a particular eye compartment are likely to be selectively expressed in that compartment, we compared our gene expression signatures with genetic mapping studies to identify candidate genes for diseases affecting the cornea, lens, and retina. Conclusion Through genome-scale gene expression profiling, we were able to discover distinct gene expression 'signatures' for each eye compartment and identified candidate disease genes that can serve as a reference database for investigating the physiology and pathophysiology of the eye. PMID:16168081

  18. Uterus human leucocyte antigen expression in the perspective of transplantation.

    PubMed

    Gauthier, Tristan; Filloux, Matthieu; Guillaudeau, Angelique; Essig, Marie; Bibes, Romain; Pacha, Adam Fodil; Piver, Pascal; Aubard, Yves; Marquet, Pierre; Drouet, Mireille

    2016-12-01

    To describe class I and II human leucocyte antigen (HLA) expression using different uterine tissues in the perspective of uterus transplantation. Human uterine tissues were obtained from 12 women who had undergone hysterectomy for the treatment of benign disease. HLA class I and HLA-antigen D related (DR) expression were assessed via immunochemistry. HLA class I expression in the uterus was compared with expression in other organs and tissues, including kidney and myocardium samples. HLA class I expression was strong in the endometrial glands and mild in the myometrium. Staining of endometrial glands was similar to glomerular staining in the kidney. The myometrium seems to express HLA class I similarly to hepatocytes and myocardial cells. HLA class I expression in the uterus did not differ in younger or post-menopausal women. HLA-DR was expressed in the endometrial glands, but not in the myometrium. A lack of HLA-DR expression seemed to be correlated with cell proliferation. HLA expression in the endometrium and myometrium is different. The endometrium should be the major target of alloreactive response. As for other transplanted organs, assessment of HLA unacceptable antigens and multiple immunosuppressive treatments is necessary in uterus transplantation. © 2016 Japan Society of Obstetrics and Gynecology.

  19. Corticosterone inhibits normal and FSH-induced testicular recrudescence in the lizard, Mabuya carinata.

    PubMed

    Yajurvedi, H N; Nijagal, B S

    2000-12-01

    Administration (ip) of 1, 10, or 20 microg corticosterone (alternate days for 30 days) to adult male Mabuya carinata did not affect the seasonal recrudescence of spermatogenesis whereas administration of 40 microg corticosterone did result in inhibition of spermatogenesis. Further, administration of FSH (10 IU/lizard/alternate day for 30 days) during the quiescent phase of the testicular cycle stimulated spermatogenetic and steroidogenic activity of the testis as shown by significant increases in the mean number of spermatogonia, spermatocytes, and spermatids and serum levels of testosterone. In addition there were abundant spermatozoa in the lumen of the tubules in FSH-treated lizards. Administration of 10 IU FSH + 40 microg corticosterone (per lizard on alternate days for 30 days) increased the mean number of primary and secondary spermatocytes whereas the mean number of spermatids did not show significant variation compared with that of controls. Further, the mean numbers of spermatocytes and spermatids and serum levels of testosterone were significantly less when compared to those of FSH alone treated lizards. In addition, FSH-induced development of epididymis was also inhibited by corticosterone treatment. The results indicate that corticosterone inhibits FSH-induced testicular recrudescence, possibly by suppressing testosterone secretion in M. carinata.

  20. Redirecting intracellular trafficking and the secretion pattern of FSH dramatically enhances ovarian function in mice

    PubMed Central

    Wang, Huizhen; Larson, Melissa; Jablonka-Shariff, Albina; Pearl, Christopher A.; Miller, William L.; Conn, P. Michael; Boime, Irving; Kumar, T. Rajendra

    2014-01-01

    FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes. PMID:24706813

  1. Downregulation of clusterin expression in human testicular seminoma.

    PubMed

    Liu, Bianjiang; Han, Min Tang Zhijian; Zhang, Jiexiu; Lu, Pei; Li, Jie; Song, Ninghong; Wang, Zengjun; Yin, Changjun; Zhang, Wei

    2013-01-01

    Clusterin, a heterodimeric glycoprotein of approximately 80 kDa, exists extensively in human body fluids. The abnormal expression of clusterin is closely related to the occurrence, progression, and prognosis of tumors. Up to now, few studies have focused on clusterin in human testicular cancer. This study describes an extensive exploration of the presence and expression of clusterin in testicular seminoma. Tumor tissues and normal testis tissues were collected from 13 patients with testicular seminoma and 16 patients undergoing surgical castration for prostate cancer. Real-time polymerase chain reaction (PCR) was performed to detect the expression difference of clusterin mRNA between testicular seminoma and normal testis. Western blot and immunohistochemical analysis were performed to detect the presence and expression difference of clusterin protein between two groups. Real-time PCR showed the expression of clusterin mRNA in testicular seminoma to be significantly lower than in normal testis (only 13% relative quantification). Western blot analysis indicated marked reductions in the expression of clusterin protein in testicular seminoma. Similar results were observed upon immunohistochemical analysis. In testicular seminoma and normal testis, clusterin exists in its heterodimeric secretory isoform. Clusterin expression is significantly lower in testicular seminoma than in normal testis. This is the first comprehensive study of the presence and expression of clusterin in human testicular cancer. © 2013 S. Karger AG, Basel

  2. Conserved Expression Signatures between Medaka and Human Pigment Cell Tumors

    PubMed Central

    Schartl, Manfred; Kneitz, Susanne; Wilde, Brigitta; Wagner, Toni; Henkel, Christiaan V.; Spaink, Herman P.; Meierjohann, Svenja

    2012-01-01

    Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules. PMID:22693581

  3. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  4. Ovotoxic Effects of Galactose Involve Attenuation of Follicle-Stimulating Hormone Bioactivity and Up-Regulation of Granulosa Cell p53 Expression

    PubMed Central

    Banerjee, Sayani; Chakraborty, Pratip; Saha, Piyali; Bandyopadhyay, Soma Aditya; Banerjee, Sutapa; Kabir, Syed N.

    2012-01-01

    Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented

  5. Expression of intermediate filaments at muscle insertions in human fetuses

    PubMed Central

    Abe, Shinichi; Rhee, Sun-ki; Osonoi, Makoto; Nakamura, Takuo; Cho, Baik Hwan; Murakami, Gen; Ide, Yoshinobu

    2010-01-01

    Desmin and vimentin are intermediate filaments that play crucial roles in the maturation, maintenance and recovery of muscle fibers and mesenchymal cells. The expression of these proteins has not been investigated extensively in human fetuses. In the present study, we examined the immunohistochemical expression of intermediate filaments in skeletal muscles of the head, neck and thorax in 12 mid-term human fetuses at 9–18 weeks of gestation. We also used immunohistochemistry to localize the expression of the myosin heavy chain and silver impregnation to identify the fetal endomysium. Expression of desmin and vimentin was already detectable in intercostal muscle at 9 weeks, especially at sites of muscle attachment to the perichondrium. At this stage, myosin heavy chain was expressed throughout the muscle fibers and the endomysium had already developed. Beginning with punctate expression, the positive areas became diffusely distributed in the muscle fibers. At 15–18 weeks, intermediate filament proteins were extensively expressed in all of the muscles examined. Expression at the bone–muscle interface was continuous with expression along the intramuscular tendon fibres. These results suggest that the development of intermediate filaments begins in areas of mechanical stress due to early muscle contraction. Their initially punctate distribution, as observed here, probably corresponds to the earliest stage of fetal enthesis formation. PMID:20500537

  6. The expression of ADAMTS13 in human microvascular endothelial cells.

    PubMed

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells.

  7. Human Empathy, Personality and Experience Affect the Emotion Ratings of Dog and Human Facial Expressions

    PubMed Central

    Kujala, Miiamaaria V.; Somppi, Sanni; Jokela, Markus; Vainio, Outi; Parkkonen, Lauri

    2017-01-01

    Facial expressions are important for humans in communicating emotions to the conspecifics and enhancing interpersonal understanding. Many muscles producing facial expressions in humans are also found in domestic dogs, but little is known about how humans perceive dog facial expressions, and which psychological factors influence people’s perceptions. Here, we asked 34 observers to rate the valence, arousal, and the six basic emotions (happiness, sadness, surprise, disgust, fear, and anger/aggressiveness) from images of human and dog faces with Pleasant, Neutral and Threatening expressions. We investigated how the subjects’ personality (the Big Five Inventory), empathy (Interpersonal Reactivity Index) and experience of dog behavior affect the ratings of dog and human faces. Ratings of both species followed similar general patterns: human subjects classified dog facial expressions from pleasant to threatening very similarly to human facial expressions. Subjects with higher emotional empathy evaluated Threatening faces of both species as more negative in valence and higher in anger/aggressiveness. More empathetic subjects also rated the happiness of Pleasant humans but not dogs higher, and they were quicker in their valence judgments of Pleasant human, Threatening human and Threatening dog faces. Experience with dogs correlated positively with ratings of Pleasant and Neutral dog faces. Personality also had a minor effect on the ratings of Pleasant and Neutral faces in both species. The results imply that humans perceive human and dog facial expression in a similar manner, and the perception of both species is influenced by psychological factors of the evaluators. Especially empathy affects both the speed and intensity of rating dogs’ emotional facial expressions. PMID:28114335

  8. Human Empathy, Personality and Experience Affect the Emotion Ratings of Dog and Human Facial Expressions.

    PubMed

    Kujala, Miiamaaria V; Somppi, Sanni; Jokela, Markus; Vainio, Outi; Parkkonen, Lauri

    2017-01-01

    Facial expressions are important for humans in communicating emotions to the conspecifics and enhancing interpersonal understanding. Many muscles producing facial expressions in humans are also found in domestic dogs, but little is known about how humans perceive dog facial expressions, and which psychological factors influence people's perceptions. Here, we asked 34 observers to rate the valence, arousal, and the six basic emotions (happiness, sadness, surprise, disgust, fear, and anger/aggressiveness) from images of human and dog faces with Pleasant, Neutral and Threatening expressions. We investigated how the subjects' personality (the Big Five Inventory), empathy (Interpersonal Reactivity Index) and experience of dog behavior affect the ratings of dog and human faces. Ratings of both species followed similar general patterns: human subjects classified dog facial expressions from pleasant to threatening very similarly to human facial expressions. Subjects with higher emotional empathy evaluated Threatening faces of both species as more negative in valence and higher in anger/aggressiveness. More empathetic subjects also rated the happiness of Pleasant humans but not dogs higher, and they were quicker in their valence judgments of Pleasant human, Threatening human and Threatening dog faces. Experience with dogs correlated positively with ratings of Pleasant and Neutral dog faces. Personality also had a minor effect on the ratings of Pleasant and Neutral faces in both species. The results imply that humans perceive human and dog facial expression in a similar manner, and the perception of both species is influenced by psychological factors of the evaluators. Especially empathy affects both the speed and intensity of rating dogs' emotional facial expressions.

  9. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  10. A novel SGLT is expressed in the human kidney.

    PubMed

    Kothinti, Rajendra K; Blodgett, Amy B; North, Paula E; Roman, Richard J; Tabatabai, Niloofar M

    2012-09-05

    Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury.

  11. A novel SGLT is expressed in the human kidney

    PubMed Central

    Kothinti, Rajendra K.; Blodgett, Amy B.; North, Paula E.; Roman, Richard J.; Tabatabai, Niloofar M.

    2013-01-01

    Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50 μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50 μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury. PMID:22766068

  12. Comparison by three different radioimmunoassay systems of the polymorphism of plasma FSH in mares in various reproductive states.

    PubMed

    Alexander, S L; Irvine, C H; Turner, J E

    1987-01-01

    FSH was measured in the pituitary, and in pituitary venous and jugular blood collected at frequent intervals from mares in various reproductive states, using 3 validated and highly specific radioimmunoassay systems based on different antibodies, 'o', 'h' and 'e'. In the pituitary, 4 forms of FSH were found which differed in isoelectric point and relative potency in the 3 assays. In jugular blood, mean FSH concentrations and short-term patterns depended on the assay used and the reproductive state of the mare. In pituitary venous blood, although FSH concentrations were greatly elevated above jugular values, the relationship amongst the 3 assays was similar to that in jugular blood. However, the pulsatility of FSH secretion at oestrus (1-2 pulses per h in 4 of 5 mares) could be observed only in pituitary blood. When the rate of folliculogenesis was slow (acyclicity, oestrus), FSH concentrations measured in assay 'o' were relatively high and showed smaller fluctuations than in assays 'h' and 'e'. By contrast, when folliculogenesis was rapid (transitional phase, dioestrus), FSH tended to be lowest in assay 'o' and pulses had a similar magnitude in all assays. These results suggest that plasma FSH is polymorphic and that changes in the circulating form may have functional importance. Furthermore, since FSH polymorphism affects immunoactivity, the choice of a radioimmunoassay for clinical or research use is difficult without further FSH structure-function studies to determine which form of the hormone should be measured in a given situation.

  13. Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep.

    PubMed

    Ammoun, I; Encinas, T; Veiga-Lopez, A; Ros, J M; Contreras, I; Gonzalez-Añover, P; Cocero, M J; McNeilly, A S; Gonzalez-Bulnes, A

    2006-09-01

    Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of

  14. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Developmental expression of mucin genes in the human gastrointestinal system

    PubMed Central

    Reid, C; Harris, A

    1998-01-01

    Background and aims—Mucin glycoproteins play a key role in the normal function of the epithelium lining the gastrointestinal tract. The expression of mucin genes, MUC 3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues was examined to establish the localisation and age of onset of expression of each mucin gene during human development. 
Methods—Mucin gene expression was assayed by mRNA in situ hybridisation. 
Results—Expression of MUC3 was detected in the small intestine and colon from 13 weeks gestation onwards and at low levels in the main pancreatic duct at 13 weeks only. MUC4 expression was seen at a low level in the colonic epithelium from 13 weeks of gestation but not elsewhere in the gastrointestinal tract. MUC5AC mRNA was detected in the colon at 17 weeks and at high levels in the stomach at 23 weeks. MUC6 transcripts were evident in the pancreatic ducts from 13 weeks of gestation and at high levels in the stomach at 23 weeks. MUC5B, MUC7, and MUC8 transcripts were not detected. 
Conclusions—Mucin genes are expressed from the early mid-trimester of gestation in the developing human fetal gastrointestinal tract. 

 Keywords: mucin; developmental expression; gastrointestinal tract PMID:9536947

  16. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    PubMed Central

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  17. Sequence and expression analysis of gaps in human chromosome 20.

    PubMed

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan; El-Schich, Zahra; Bak, Mads; Hansen, Claus; Papadopoulos, Nickolas; Josefsen, Knud; Nielsen, Henrik; Gorodkin, Jan; Tommerup, Niels; Silahtaroglu, Asli

    2012-08-01

    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and/or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ∼ 99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum. One of these CpG islands was differentially methylated and paternally hypermethylated. We found all chr 20 gaps to comprise structured non-coding RNAs (ncRNAs) and to be conserved in primates. We verified expression for 13 candidate ncRNAs, some of which showed tissue specificity. Four ncRNAs expressed within the gap at DLGAP4 show elevated expression in the human brain. Our data suggest that unfinished human genome gaps are likely to comprise numerous functional elements.

  18. Sequence and expression analysis of gaps in human chromosome 20

    PubMed Central

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan; El-schich, Zahra; Bak, Mads; Hansen, Claus; Papadopoulos, Nickolas; Josefsen, Knud; Nielsen, Henrik; Gorodkin, Jan; Tommerup, Niels; Silahtaroglu, Asli

    2012-01-01

    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and/or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ∼99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum. One of these CpG islands was differentially methylated and paternally hypermethylated. We found all chr 20 gaps to comprise structured non-coding RNAs (ncRNAs) and to be conserved in primates. We verified expression for 13 candidate ncRNAs, some of which showed tissue specificity. Four ncRNAs expressed within the gap at DLGAP4 show elevated expression in the human brain. Our data suggest that unfinished human genome gaps are likely to comprise numerous functional elements. PMID:22510267

  19. 200 THE USE OF LONG-ACTING FSH-MAP5 IN SHEEP SUPEROVULATION PROGRAMS.

    PubMed

    Fry, R

    2016-01-01

    The administration of sustained-release FSH-MAP5 in a 2-injection protocol has been shown to be as effective as a multiple-FSH injection protocol in inducing superovulation in cattle (Bo and Mapletoft 2014 Theriogenology 81, 38) and in sheep (Fry et al. 2016 Reprod. Fertil. Dev. 28, 250); however, the effect on embryo quality in the latter experiment was unclear. The following experiment further investigated the effect of FSH-MAP5 on ovulation rate and embryo quality in a sheep multiple-ovulation embryo transfer (MOET) program conducted in the breeding season. Two hundred sixteen Dohne merino ewes received a 12-day CIDR-S device containing 0.33g of progesterone (Zoetis, Florham Park, NJ, USA) plus 200mg of FSH IM (Folltropin-V; Vetoquinol, Belleville, Canada) and 400IU of eCG IM (Pregnecol: Vetoquinol) in 4 treatment groups. Group 1 (n=51) received 7 injections (a.m., p.m.) of FSH in saline (2.5, 2.0, 1.5, 1.5, 1.0, 1.0, and 0.5mL) starting 2.5 days before CIDR withdrawal and 400IU of eCG in saline at the time of the first FSH injection. Group 2 (n=53) received 6 injections (a.m., p.m.) of FSH in saline (3.0, 2.0, 1.5, 1.5, 1.0, and 1.0mL) starting 2.5 days before CIDR withdrawal and 400IU of eCG in saline at CIDR withdrawal. Group 3 (n=56) received 3.3mL of FSH in hyaluronan (50mg of MAP5, Vetoquinol) and 400IU of eCG in saline 2.5 days before CIDR withdrawal and 1.7mL of FSH-MAP5 at 0.5 days before CIDR withdrawal. Group 4 (n=56) received 3.3mL of FSH-MAP5 at 2.5 days before CIDR withdrawal, 1.7mL of FSH-MAP5 at 0.5 days before CIDR withdrawal, and 400IU of eCG in saline at CIDR withdrawal. Ewes were inseminated with semen collected and pooled from 5 rams at 36 to 40h after CIDR withdrawal. Donor ewes were slaughtered 6 days after AI, corpora lutea were counted on the ovary, and ova/embryos were collected. Data for corpora lutea, total ova/embryo, and transferable embryo was analysed by the Kruskal-Wallis test and differences between groups were determined by the

  20. Monoallelic expression of the human FOXP2 speech gene.

    PubMed

    Adegbola, Abidemi A; Cox, Gerald F; Bradshaw, Elizabeth M; Hafler, David A; Gimelbrant, Alexander; Chess, Andrew

    2015-06-02

    The recent descriptions of widespread random monoallelic expression (RMAE) of genes distributed throughout the autosomal genome indicate that there are more genes subject to RMAE on autosomes than the number of genes on the X chromosome where X-inactivation dictates RMAE of X-linked genes. Several of the autosomal genes that undergo RMAE have independently been implicated in human Mendelian disorders. Thus, parsing the relationship between allele-specific expression of these genes and disease is of interest. Mutations in the human forkhead box P2 gene, FOXP2, cause developmental verbal dyspraxia with profound speech and language deficits. Here, we show that the human FOXP2 gene undergoes RMAE. Studying an individual with developmental verbal dyspraxia, we identify a deletion 3 Mb away from the FOXP2 gene, which impacts FOXP2 gene expression in cis. Together these data suggest the intriguing possibility that RMAE impacts the haploinsufficiency phenotypes observed for FOXP2 mutations.

  1. Monoallelic expression of the human FOXP2 speech gene

    PubMed Central

    Adegbola, Abidemi A.; Cox, Gerald F.; Bradshaw, Elizabeth M.; Hafler, David A.; Gimelbrant, Alexander; Chess, Andrew

    2015-01-01

    The recent descriptions of widespread random monoallelic expression (RMAE) of genes distributed throughout the autosomal genome indicate that there are more genes subject to RMAE on autosomes than the number of genes on the X chromosome where X-inactivation dictates RMAE of X-linked genes. Several of the autosomal genes that undergo RMAE have independently been implicated in human Mendelian disorders. Thus, parsing the relationship between allele-specific expression of these genes and disease is of interest. Mutations in the human forkhead box P2 gene, FOXP2, cause developmental verbal dyspraxia with profound speech and language deficits. Here, we show that the human FOXP2 gene undergoes RMAE. Studying an individual with developmental verbal dyspraxia, we identify a deletion 3 Mb away from the FOXP2 gene, which impacts FOXP2 gene expression in cis. Together these data suggest the intriguing possibility that RMAE impacts the haploinsufficiency phenotypes observed for FOXP2 mutations. PMID:25422445

  2. Expression of human adenosine deaminase in murine hematopoietic cells.

    PubMed Central

    Belmont, J W; MacGregor, G R; Wager-Smith, K; Fletcher, F A; Moore, K A; Hawkins, D; Villalon, D; Chang, S M; Caskey, C T

    1988-01-01

    Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells. Images PMID:3072474

  3. Primary gonadal failure in men selectively amplifies the mass of follicle stimulating hormone (FSH) secreted per burst and increases the disorderliness of FSH release patterns: reversibility with testosterone replacement.

    PubMed

    Veldhuis, J D; Iranmanesh, A; Urban, R J

    1997-10-01

    The neuroendocrine mechanisms by which primary gonadal failure in men increases mean serum FSH concentrations (castration-like response) are not known. To investigate the testosterone-dependent mechanisms of the FSH castration response: (i) blood was sampled at 10-min intervals for 24 h for later FSH assay in seven normal middle-aged men and in six patients with primary testicular failure, during testosterone withdrawal and after 6 weeks of parenteral testosterone replacement; (ii) using a specific two-site IRMA, serum FSH concentrations were measured, since this assay correlates well with an in-vitro Sertoli cell bioassay; (iii) multiparameter deconvolution analysis was then applied to estimate the frequency, amplitude, duration, and mass of underlying FSH secretory bursts, and the half-life of endogenous FSH, and (iv) approximate entropy was calculated to quantify the relative orderliness of FSH release over 24 h. Mean (+/- SEM) 24-h serum FSH concentrations were 3.9 +/- 0.8 IU/L in control subjects and 39 +/- 10 IU/L in unreplaced hypogonadal patients (p = 0.034). Deconvolution analysis revealed similar estimated mean FSH half-lives of 346 +/- 40 min (control) and 321 +/- 47 min (untreated patients), and indistinguishable FSH secretory burst frequencies, namely, 20 +/- 0.95 (normal) and 21 +/- 1.3 (patients) pulses per 24 h. In contrast, the daily production rate of FSH was markedly increased in testosterone-withdrawn hypogonadal men at 117 +/- 25 vs. 9.3 +/- 1.8 IU/L/day (control) (p < 0.01). This was due to a 10-fold higher calculated maximal rate (amplitude) of FSH secretion achieved within each FSH release episode (normal 0.078 +/- 0.02 vs. gonadal failure 0.74 +/- 0.087 IU/L/min, p < 0.01), yielding a 10-fold increase in the mass of FSH secreted per burst (control 0.53 +/- 0.06 vs. patients 5.3 +/- 0.81 IU/L, p < 0.01). In contrast, the mean half-duration of FSH secretory bursts was unaltered in unreplaced hypogonadal men at 8.2 +/- 2.2 min (control) vs. 7

  4. Four zona pellucida glycoproteins are expressed in the human.

    PubMed

    Lefièvre, L; Conner, S J; Salpekar, A; Olufowobi, O; Ashton, P; Pavlovic, B; Lenton, W; Afnan, M; Brewis, I A; Monk, M; Hughes, D C; Barratt, C L R

    2004-07-01

    The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model

  5. Characterization of the Olfactory Receptors Expressed in Human Spermatozoa

    PubMed Central

    Flegel, Caroline; Vogel, Felix; Hofreuter, Adrian; Schreiner, Benjamin S. P.; Osthold, Sandra; Veitinger, Sophie; Becker, Christian; Brockmeyer, Norbert H.; Muschol, Michael; Wennemuth, Gunther; Altmüller, Janine; Hatt, Hanns; Gisselmann, Günter

    2016-01-01

    The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa. PMID:26779489

  6. Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.

    PubMed

    Chiba, K; Kobayashi, H; Wakabayashi, K

    1997-04-01

    A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography. In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDA, respectively. The purified hormones showed two bands corresponding to alpha (20 kDa) and beta subunits (cLH beta: 16 kDa, cFSH beta: 22 kDa, cTSH beta: 16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Cross-contamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

  7. Analysis of recombinant human tumour necrosis factor-alpha-induced CD4 expression on human eosinophils.

    PubMed Central

    Hossain, M; Okubo, Y; Horie, S; Sekiguchi, M

    1996-01-01

    We examined the hypothesis that one of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), could induce expression of the adhesion molecule CD4 on human eosinophils. We further examined the effector function of CD4 and the mechanisms regulating CD4 expression. Human eosinophils were cultured with various concentrations of recombinant human TNF-alpha (rhTNF-alpha) with or without various drugs for 24 hr. After culture, eosinophils were stained for CD4 using a monoclonal antibody and then analysed by flow cytometry. Eosinophil-derived neurotoxin (EDN) release as eosinophil degranulation was examined by cross-linking of CD4 on eosinophils. The rhTNF-alpha induced CD4 expression on human eosinophils in a dose- and time-dependent fashion; rhTNF-alpha-induced CD4 expression was significantly inhibited by 10(-6) M cycloheximide, 10(-8) M dexamethasone, or 10(-6) M herbimycin A. Recombinant human interferon-gamma inhibited rhTNF-alpha-induced CD4 expression in a dose-dependent manner. However, cross-linking of CD4 on eosinophils did not evoke EDN release, suggesting that newly expressed CD4 molecules on human eosinophils do not play any role in triggering degranulation. Our data indicate that TNF-alpha-induced CD4 expression on human eosinophils is dependent on protein synthesis and may be dependent on tyrosine kinase activity. PMID:8690465

  8. DNMT3B7 expression related to MENT expression and its promoter methylation in human lymphomas.

    PubMed

    Alkebsi, Lobna; Handa, Hiroshi; Sasaki, Yoshiko; Osaki, Yohei; Yanagisawa, Kunio; Ogawa, Yoshiaki; Yokohama, Akihiko; Hattori, Hikaru; Koiso, Hiromi; Saitoh, Takayuki; Mitsui, Takeki; Tsukamoto, Norifumi; Nojima, Yoshihisa; Murakami, Hirokazu

    2013-12-01

    DNA methyltransferase (DNMT) 3B7 is the most expressed DNMT3B splice variant. It was reported that the loss of DNMT3B function led to overexpression of the MEthylated in Normal Thymocyes (MENT) and accelerated mouse lymphomagenesis. We investigated the DNMT3B7 expression and its relationship to MENT expression and promoter methylation in human lymphomas. DNMT3B7 and MENT expression were significantly (p<0.0001, p<0.01) higher in lymphomas than in non-malignant. Expression of DNMT3B7 and MENT were associated with MENT promoter hypomethylation. DNMT3B7 overexpression might interfere with the normal DNA methylation mechanism required for silencing the MENT proto-oncogene, and may accelerate human lymphomagenesis.

  9. Right ventricular long noncoding RNA expression in human heart failure

    PubMed Central

    Guo, Yan; Su, Yan Ru; Clark, Travis; Brittain, Evan; Absi, Tarek; Maltais, Simon; Hemnes, Anna

    2015-01-01

    Abstract The expression of long noncoding RNAs (lncRNAs) in human heart failure (HF) has not been widely studied. Using RNA sequencing (RNA-Seq), we compared lncRNA expression in 22 explanted human HF hearts with lncRNA expression in 5 unused donor human hearts. We used Cufflinks to identify isoforms and DESeq to identify differentially expressed genes. We identified the noncoding RNAs by cross-reference to Ensembl release 73 (Genome Reference Consortium human genome build 37) and explored possible functional roles using a variety of online tools. In HF hearts, RNA-Seq identified 84,793 total messenger RNA coding and noncoding different transcripts, including 13,019 protein-coding genes, 2,085 total lncRNA genes, and 1,064 pseudogenes. By Ensembl noncoding RNA categories, there were 48 lncRNAs, 27 pseudogenes, and 30 antisense RNAs for a total of 105 differentially expressed lncRNAs in HF hearts. Compared with donor hearts, HF hearts exhibited differential expression of 7.7% of protein-coding genes, 3.7% of lncRNAs (including pseudogenes), and 2.5% of pseudogenes. There were not consistent correlations between antisense lncRNAs and parent genes and between pseudogenes and parent genes, implying differential regulation of expression. Exploratory in silico functional analyses using online tools suggested a variety of possible lncRNA regulatory roles. By providing a comprehensive profile of right ventricular polyadenylated messenger RNA transcriptome in HF, RNA-Seq provides an inventory of differentially expressed lncRNAs, including antisense transcripts and pseudogenes, for future mechanistic study. PMID:25992278

  10. Epithelin/Granulin Precursor Expression in Human Breast Carcinoma

    DTIC Science & Technology

    1998-09-01

    presented in this paper summarized here show that PCDGF mRNA and protein is expressed in estrogen-dependent human breast carcinoma cell lines MCF-7 and...T47D. Treatment of human breast carcinoma cell lines MCF-7 and T47D with estradiol stimulated PCDGF mRNA and protein expression in a dose (5-fold...and protein were very low in the non-tumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic

  11. Expression of growth hormone receptor in the human brain.

    PubMed

    Castro, J R; Costoya, J A; Gallego, R; Prieto, A; Arce, V M; Señarís, R

    2000-03-10

    This study was designed to investigate the presence of growth hormone receptor (GHR) expression in the human brain tissue, both normal and tumoral, as well as in the human glioblastoma cell line U87MG. Reverse transcription-polymerase chain reaction revealed the presence of GHR mRNA in all brain samples investigated and in U87MG cells. GHR immunoreactivity was also detected in this cell line using both immunocytochemistry and western blotting. All together, our data demonstrate the existence of GHR expression within the central nervous system (CNS), thus supporting a possible role for GH in the CNS physiology.

  12. Expression of nNOS in the human larynx.

    PubMed

    Şelaru, Mircea; Rusu, Mugurel Constantin; Jianu, Adelina Maria

    2015-09-01

    Although intrinsic laryngeal neurons and ganglia have been studied in various species, they have been overlooked in humans. We aimed to investigate the presence of intrinsic laryngeal neurons in humans and, if present, to analyze their neuronal nitric oxide synthase (nNOS) expression. An immunohistochemical study using anti-nNOS antibodies was performed on samples obtained from four cadavers. Intrinsic laryngeal nNOS+ neurons were assessed in the submucosal layer, but nNOS+ nerves were found in all histological layers of the larynx. nNOS expression was also found in striated muscle fibers of larynx. This might reveal the anatomical basis of an upwards extension of the nonadrenergic noncholinergic system in human airways, but further experiments are needed to assess an exact role of NO influence on neural transmission and muscular functions of human larynx.

  13. Importance of the brow in facial expressiveness during human communication.

    PubMed

    Neely, John Gail; Lisker, Paul; Drapekin, Jesse

    2014-03-01

    The objective of this study was to evaluate laterality and upper/lower face dominance of expressiveness during prescribed speech using a unique validated image subtraction system capable of sensitive and reliable measurement of facial surface deformation. Observations and experiments of central control of facial expressions during speech and social utterances in humans and animals suggest that the right mouth moves more than the left during nonemotional speech. However, proficient lip readers seem to attend to the whole face to interpret meaning from expressed facial cues, also implicating a horizontal (upper face-lower face) axis. Prospective experimental design. Experimental maneuver: recited speech. image-subtraction strength-duration curve amplitude. Thirty normal human adults were evaluated during memorized nonemotional recitation of 2 short sentences. Facial movements were assessed using a video-image subtractions system capable of simultaneously measuring upper and lower specific areas of each hemiface. The results demonstrate both axes influence facial expressiveness in human communication; however, the horizontal axis (upper versus lower face) would appear dominant, especially during what would appear to be spontaneous breakthrough unplanned expressiveness. These data are congruent with the concept that the left cerebral hemisphere has control over nonemotionally stimulated speech; however, the multisynaptic brainstem extrapyramidal pathways may override hemiface laterality and preferentially take control of the upper face. Additionally, these data demonstrate the importance of the often-ignored brow in facial expressiveness. Experimental study. EBM levels not applicable.

  14. Genetic effects on gene expression across human tissues.

    PubMed

    Battle, Alexis; Brown, Christopher D; Engelhardt, Barbara E; Montgomery, Stephen B

    2017-10-11

    Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease.

  15. Notch receptor expression in human brain arteriovenous malformations.

    PubMed

    Hill-Felberg, Sandra; Wu, Hope Hueizhi; Toms, Steven A; Dehdashti, Amir R

    2015-08-01

    The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1-4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.

  16. Early effects of equine FSH (eFSH) treatment on hormonal and reproductive parameters in mares intended to carry their own pregnancy.

    PubMed

    Raz, Tal; Gray, Allister; Hunter, Barbara; Card, Claire

    2009-10-01

    Superovulatory treatment may potentially increase the embryo recovery rate and the per-cycle pregnancy rate in normal or subfertile mares that are managed properly. However, some studies suggest a possible negative effect of superovulatory treatment on ovarian follicular maturation and embryo viability. Objectives of the present study were to investigate the early effects of eFSH treatment in reproductively normal mares in terms of: folliculogenesis, pregnancy rate, early embryonic development, reproductive tract parameters (tone and edema), and serum estradiol-17beta and progesterone concentrations. Reproductively sound mares (n=26) were evaluated daily by transrectal palpation and ultrasonography. Five days after spontaneous ovulation, mares were randomly assigned to one of two treatment groups. In the eFSH group, mares (n=16 estrous cycles) were administered eFSH twice daily; beginning when a follicle > or =20mm was detected, and continuing until at least one follicle reached a diameter of > or =35 mm. PGF2alpha was administered 2 days following initiation of eFSH therapy, and hCG was administered approximately 36h after cessation of eFSH therapy. In the control group, mares (n=26 estrous cycles) were administered PGF2alpha 7 days after spontaneous ovulation, and hCG when a follicle > or =35 mm was detected. All mares were bred with fresh semen, monitored for ovulation (Day 0), and evaluated for pregnancy on Days 11-16. Serum estradiol-17beta and progesterone concentrations were analyzed using radioimmunoassay on the Day of hCG administration, and Days 8, 11 and 16. Mares treated with eFSH had more follicles > or =30 mm at the time of hCG administration (2.6+/-0.4 compared with 1.1+/-0.1; P<0.01), and more ovulations (2.3+/-0.5 compared with 1.1+/-0.3; P<0.01). However, pregnancy rates were not significantly different between groups (50%; 8/16 compared with 62%; 16/26). Mean overall daily growth rate of embryonic vesicles from Day 11 to 16 was not statistically

  17. Expression of human hyaluronan synthases in response to external stimuli.

    PubMed Central

    Jacobson, A; Brinck, J; Briskin, M J; Spicer, A P; Heldin, P

    2000-01-01

    In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins. PMID:10794710

  18. The mirror RNA expression pattern in human tissues

    PubMed Central

    Bythwood, Tameka N.; Xu, Wei; Li, Wenzhi; Rao, Weinian; Li, Qiling; Xue, Xue; Richards, Jendai; Ma, Li; Song, Qing

    2017-01-01

    It has been realized in recent years that non-coding RNAs are playing important roles in genome functions and human diseases. Here we developed a new technology and observed a new pattern of gene expression. We observed that over 72% of RNAs in human genome are expressed in forward-reverse pairs, just like mirror images of each other between forward expression and reverse expression; the overview showed that it cannot be simply described as transcript overlapping, so we designated it as mirror expression. Furthermore, we found that the mirror expression is gene-specific and tissue-specific, and less common in the proximal promoter regions. The size of the shadows varies between different genes, different tissues and different classes. The shadow expression is most significant in the Alu element, it was also observed among L1, Simple Repeats and LTR elements, but rare in other repeats such as low-complexity, LINE/L2, DNA and MIRs. Although there is no evidence yet about the relationship of this mirror pattern and double-strand RNA (dsRNA), this new striking pattern provides a new clue and a new direction to unveil the role of RNAs in the genome functions and diseases.

  19. Expression of aquaporin isoforms during human and mouse tooth development.

    PubMed

    Felszeghy, S; Módis, L; Németh, P; Nagy, G; Zelles, T; Agre, P; Laurikkala, J; Fejerskov, O; Thesleff, I; Nielsen, S

    2004-04-01

    Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

  20. Somatic expression of LINE-1 elements in human tissues

    PubMed Central

    Belancio, Victoria P.; Roy-Engel, Astrid M.; Pochampally, Radhika R.; Deininger, Prescott

    2010-01-01

    LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated β-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging. PMID:20215437

  1. Universality and flexibility in gene expression from bacteria to human

    PubMed Central

    Ueda, Hiroki R.; Hayashi, Satoko; Matsuyama, Shinichi; Yomo, Tetsuya; Hashimoto, Seiichi; Kay, Steve A.; Hogenesch, John B.; Iino, Masamitsu

    2004-01-01

    Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from E. coli to human, where gene expression changes are proportional to their expression levels, and show that this “proportional” dynamics or “rich-travel-more” mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels. PMID:14999098

  2. Regulated Proenkephalin Expression in Human Skin and Cultured Skin Cells

    PubMed Central

    Slominski, Andrzej T.; Zmijewski, Michal A.; Zbytek, Blazej; Brozyna, Anna A.; Granese, Jackie; Pisarchik, Alexander; Szczesniewski, Andre; Tobin, Desmond J.

    2011-01-01

    Skin responds to environmental stressors via coordinated actions of the local neuroimmunoendocrine system. Although some of these responses involve opioid receptors, little is known about cutaneous proenkephalin expression, its environmental regulation, and alterations in pathology. The objective of this study was to assess regulated expression of proenkephalin in normal and pathological skin and in isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells. The proenkephalin gene and protein were expressed in skin and cultured cells, with significant expression in fibroblasts and keratinocytes. Mass spectroscopy confirmed Leu- and Met-enkephalin in skin. UVR, Toll-like receptor (TLR)4, and TLR2 agonists stimulated proenkephalin gene expression in melanocytes and keratinocytes in a time- and dose-dependent manner. In situ Met/Leu-enkephalin peptides were expressed in differentiating keratinocytes of the epidermis in the outer root sheath of the hair follicle, in myoepithelial cells of the eccrine gland, and in the basement membrane/basal lamina separating epithelial and mesenchymal components. Met/Leu-enkephalin expression was altered in pathological skin, increasing in psoriasis and decreasing in melanocytic tumors. Not only does human skin express proenkephalin, but this expression is upregulated by stressful stimuli and can be altered by pathological conditions. PMID:21191404

  3. Ceramides stimulate caspase-14 expression in human keratinocytes

    PubMed Central

    Jiang, Yan J.; Kim, Peggy; Uchida, Yoshikazu; Elias, Peter M.; Bikle, Daniel D.; Grunfeld, Carl; Feingold, Kenneth R.

    2014-01-01

    Caspase-14 is an enzyme that is expressed predominantly in cornifying epithelia and catalyses the degradation of profilaggrin. Additionally, caspase-14 plays an important role in the terminal differentiation of keratinocytes. However, how caspase-14 expression is regulated remains largely unknown. Here we demonstrate that ceramides (C2-Cer and C6-Cer), but not other sphingolipids (C8-glucosylceramides, sphinganine, sphingosine-1-phosphate or ceramide-1-phosphate), increase caspase-14 expression (mRNA and protein) in cultured human keratinocytes in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase and ceramidase increase endogenous ceramide levels and also increase caspase-14 expression, indicating an important regulatory role for ceramides and suggesting that the conversion of ceramides to other metabolites is not required. The increase in caspase-14 expression induced by ceramides is first seen at 16 h and requires new protein synthesis, suggesting that the ceramide-induced increase is likely an indirect effect. Furthermore, ceramides increase caspase-14 gene expression primarily by increasing transcription. Blocking de novo synthesis of ceramides does not affect caspase-14 expression, suggesting that basal expression is not dependent on ceramide levels. These studies show that ceramides, an important structural lipid, stimulate caspase-14 expression providing a mechanism for coordinately regulating the formation of lipid lamellar membranes with the formation of corneocytes. PMID:23362869

  4. Dynamic changes in gene expression during human trophoblast differentiation.

    PubMed

    Handwerger, Stuart; Aronow, Bruce

    2003-01-01

    The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories

  5. Expression and immunohistochemical localization of leptin in human periapical granulomas

    PubMed Central

    Martín-González, Jénifer; Carmona-Fernández, Antonio; Pérez-Pérez, Antonio; Sánchez-Jiménez, Flora; Sánchez-Margalet, Víctor

    2015-01-01

    Background Leptin, initially described as an adipocyte-derived hormone to regulate weight control, is expressed in normal and inflamed human dental pulp, being up-regulated during pulp experimental inflammation. Leptin receptor (LER) has been identified in human periapical granulomas. The aim of this study was to analyze and characterize the expression of leptin in human periapical granulomas. Material and Methods Fifteen periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their morphological categorization as periapical granulomas and gradation of the inflammatory infiltrate, they were examined by immunohistochemistry using human leptin policlonal antibodies. Leptin mRNA expression was also determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin protein was analyzed by immunoblot. Results All periapical lesions exhibited the characteristic of chronic granulomatous inflammatory process with inflammatory infiltrate grade III. Leptin+ cells were detected in 13 periapical granulomas (86.6%). The median number of Leptin+ cells in periapical granulomas was 1.70 (0.00-7.4). Amongst the inflammatory cells in the periapical granulomas, only macrophages were reactive to leptin antibodies. Western blot analysis revealed the presence in all samples of a protein with apparent molecular weight of approximately 16 kDa, corresponding to the estimated molecular weights of leptin. The expression of leptin mRNA was confirmed by qRT-PCR analysis and the size of the amplified fragment (296 bp for leptin and 194 bp for cyclophilin) was assessed by agarose gel electrophoresis. Conclusions For the first time, it has been demonstrated that human periapical granuloma expresses the adipokine leptin. Key words: Apical granuloma, dental pulp, endodontics, leptin, leptin receptor, immune system, immunohistochemistry, periapical inflammatory response. PMID:25662559

  6. Effects of photoperiod and hypothalamic knife cuts on the timing of FSH surges in hamsters.

    PubMed

    Badura, L L; Sisk, C L; Nunez, A A

    1991-02-01

    The timing of the proestrous surge of follicle-stimulating hormone (FSH) was examined in female hamsters with hypothalamic knife cuts that prevented reproductive responses to photoperiod. All animals received either a horizontal knife cut aimed between the suprachiasmatic nuclei (SCN) and the paraventricular nuclei (PVN), or sham surgery, and were housed in long (16 h of light/24 h) or short (6 h of light/24 h) photoperiods. Following exposure to either photo-period for 11-12 weeks, a subset of the animals was fitted with an indwelling jugular cannula. Blood samples were taken hourly over a 24-h period and plasma levels of FSH were determined by RIA. Knife cuts placed ventral to or through the ventral portions of the PVN prevented short day-induced anestrus. On the day of proestrus, peak elevations of FSH in cycling animals with knife cuts in both photoperiods, as well as in sham-operated females in long days, occurred 4-5 h before lights out. In contrast, sham-operated anestrous females in short days showed peak elevations of FSH approximately 3-4 h after lights out. The present results support the view that neural connections between the SCN and the PVN mediate the effects of short days on reproductive physiology, including changes in the timing of the FSH surge.

  7. Endometrial CXCL13 expression is cycle regulated in humans and aberrantly expressed in humans and Rhesus macaques with endometriosis.

    PubMed

    Franasiak, Jason M; Burns, Katherine A; Slayden, Ov; Yuan, Lingwen; Fritz, Marc A; Korach, Kenneth S; Lessey, Bruce A; Young, Steven L

    2015-04-01

    C-X-C ligand 13 (CXCL13), a regulator of mucosal immunity, is secreted by human endometrial epithelium and may be involved in embryo implantation. However, cyclic expression of human endometrial CXCL13 in health and disease is not well studied. This study examines cycle stage-specific endometrial CXCL13 expression in normal humans when compared to those with biopsy-confirmed, stage 1 to 4 endometriosis using real-time reverse transcriptase, real-time polymerase chain reaction and immunohistochemistry. Eutopic endometrial CXCL13 expression was also compared between normal, control Rhesus macaques, and macaques with advanced endometriosis. In healthy women, CXLC13 messenger RNA expression was minimal in the proliferative phase and maximal in the secretory phase. However, in the presence of endometriosis, proliferative-phase endometrial expression markedly increased in both humans and rhesus subjects (P < .05). The cross-species and cross-stage concordance suggests a pathophysiologic role for CXCL13 in endometriosis and its use as a biomarker for disease. © The Author(s) 2014.

  8. Decreased Degradation of Internalized Follicle-Stimulating Hormone Caused by Mutation of Aspartic Acid 6.30550 in a Protein Kinase-CK2 Consensus Sequence in the Third Intracellular Loop of Human Follicle-Stimulating Hormone Receptor1

    PubMed Central

    Kluetzman, Kerri S.; Thomas, Richard M.; Nechamen, Cheryl A.; Dias, James A.

    2011-01-01

    A naturally occurring mutation in follicle-stimulating hormone receptor (FSHR) gene has been reported: an amino acid change to glycine occurs at a conserved aspartic acid 550 (D550, D567, D6.30567). This residue is contained in a protein kinase-CK2 consensus site present in human FSHR (hFSHR) intracellular loop 3 (iL3). Because CK2 has been reported to play a role in trafficking of some receptors, the potential roles for CK2 and D550 in FSHR function were evaluated by generating a D550A mutation in the hFSHR. The hFSHR-D550A binds hormone similarly to WT-hFSHR when expressed in HEK293T cells. Western blot analyses showed lower levels of mature hFSHR-D550A. Maximal cAMP production of both hFSHR-D550A as well as the naturally occurring mutation hFSHR-D550G was diminished, but constitutive activity was not observed. Unexpectedly, when 125I-hFSH bound to hFSHR-D550A or hFSHR-D550G, intracellular accumulation of radiolabeled FSH was observed. Both sucrose and dominant-negative dynamin blocked internalization of radiolabeled FSH and its commensurate intracellular accumulation. Accumulation of radiolabeled FSH in cells transfected with hFSHR-D550A is due to a defect in degradation of hFSH as measured in pulse chase studies, and confocal microscopy imaging revealed that FSH accumulated in large intracellular structures. CK2 kinase activity is not required for proper degradation of internalized FSH because inhibition of CK2 kinase activity in cells expressing hFSHR did not uncouple degradation of internalized radiolabeled FSH. Additionally, the CK2 consensus site in FSHR iL3 is not required for binding because CK2alpha coimmunoprecipitated with hFSHR-D550A. Thus, mutation of D550 uncouples the link between internalization and degradation of hFSH. PMID:21270425

  9. Expression of steroidogenic enzymes in human sebaceous glands.

    PubMed

    Inoue, Takayoshi; Miki, Yasuhiro; Kakuo, Shingo; Hachiya, Akira; Kitahara, Takashi; Aiba, Setsuya; Zouboulis, Christos C; Sasano, Hironobu

    2014-09-01

    Androgens are well known to influence sebum synthesis and secretion. Various factors related to androgen biosynthesis are expressed in human sebaceous glands. In this study, immunohistochemical analysis of human skin specimens from 43 subjects indicated that various androgen-producing and -metabolizing enzymes were functionally localized to sebocytes accumulating lipid droplets and that the exclusive expression of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2 (HSD17B2)) in sebaceous glands was negatively correlated with that of peroxisome proliferator-activated receptor gamma (PPARγ (PPARG)), which also significantly changed in an age-dependent manner. We also demonstrated that the changes of 17β-HSD2 expression in human immortalized sebocytes (SZ95) influenced the expressions of sebogenesis-related factors. In addition, the overexpression of 17β-HSD2 in SZ95 significantly increased the androstenedione production and markedly decreased the amounts of testosterone and dihydrotestosterone when DHEA was added externally. On the other hand, the phosphorylation of mammalian target of rapamycin, which is well known to induce sebum secretion and the onset and/or aggravation of acne, was increased by the addition of testosterone in the presence of IGF1 in hamster sebocytes. These results all indicated that local androgen biosynthesis and metabolism in human sebaceous glands could play a pivotal role in sebum synthesis and secretion. © 2014 Society for Endocrinology.

  10. Gene expression in the aging human brain: an overview.

    PubMed

    Mohan, Adith; Mather, Karen A; Thalamuthu, Anbupalam; Baune, Bernhard T; Sachdev, Perminder S

    2016-03-01

    The review aims to provide a summary of recent developments in the study of gene expression in the aging human brain. Profiling differentially expressed genes or 'transcripts' in the human brain over the course of normal aging has provided valuable insights into the biological pathways that appear activated or suppressed in late life. Genes mediating neuroinflammation and immune system activation in particular, show significant age-related upregulation creating a state of vulnerability to neurodegenerative and neuropsychiatric disease in the aging brain. Cellular ionic dyshomeostasis and age-related decline in a host of molecular influences on synaptic efficacy may underlie neurocognitive decline in later life. Critically, these investigations have also shed light on the mobilization of protective genetic responses within the aging human brain that help determine health and disease trajectories in older age. There is growing interest in the study of pre and posttranscriptional regulators of gene expression, and the role of noncoding RNAs in particular, as mediators of the phenotypic diversity that characterizes human brain aging. Gene expression studies in healthy brain aging offer an opportunity to unravel the intricately regulated cellular underpinnings of neurocognitive aging as well as disease risk and resiliency in late life. In doing so, new avenues for early intervention in age-related neurodegenerative disease could be investigated with potentially significant implications for the development of disease-modifying therapies.

  11. Proteoglycan and collagen expression during human air conducting system development

    PubMed Central

    Godoy-Guzmán, C.; San Martin, S.; Pereda, J.

    2012-01-01

    The lung is formed from a bud that grows and divides in a dichotomous way. A bud is a new growth center which is determined by epithelial-mesenchymal interactions where proteins of the extracellular matrix (ECM) might be involved. To understand this protein participation during human lung development, we examined the expression and distribution of proteoglycans in relation to the different types of collagens during the period in which the air conducting system is installed. Using light microscopy and immunohistochemistry we evaluate the expression of collagens (I, III and VI) and proteoglycans (decorin, biglycan and lumican) between 8 to 10 weeks post fertilization and 11 to 14 weeks of gestational age of human embryo and fetus lungs. We show that decorin, lumican and all the collagen types investigated were expressed at the epithelium-mesenchymal interface, forming a sleeve around the bronchiolar ducts. In addition, biglycan was expressed in both the endothelial cells and the smooth muscle of the blood vessels. Thus, the similar distribution pattern of collagen and proteoglycans in the early developmental stages of the human lung may be closely related to the process of dichotomous division of the bronchial tree. This study provides a new insight concerning the participation of collagens and proteoglycans in the epithelial-mesenchymal interface during the period in which the air conducting system is installed in the human fetal lung. PMID:23027345

  12. Aquaporin-1 Expression and Conventional Aqueous Outflow in Human Eyes

    PubMed Central

    Stamer, W. Daniel; Chan, Darren W.H.; Conley, Shannon M.; Coons, Serena; Ethier, C. Ross

    2008-01-01

    Aquaporin channels facilitate the enhanced permeability of secretory and absorptive tissues to water. In the conventional drainage tract, aquaporin-1 is expressed but its contribution to outflow facility is unknown. The purpose of the present study was to determine the effect of elevated aquaporin-1 expression by cells of the human conventional drainage pathway on outflow facility. Using thirteen pairs of human anterior segments in organ culture, we modified aquaporin-1 protein expression in outflow cells using adenovirus encoding human aquaporin-1. Contralateral anterior segments served as controls and were transduced with adenovirus encoding beta galactosidase. By confocal immunofluorescence microscopy, we observed that inner trabecular meshwork cells from anterior segments exposed to adenovirus (via injection into the inlet tubing during perfusion) had increased aquaporin-1 protein expression compared to endogenous levels. In contrast, elevation of aquaporin-1 protein in outer meshwork cells (juxtacanalicular region) and Schlemm’s canal required transduction of adenovirus into anterior segments using retroperfusion via episcleral veins. Regardless of exposure route, outflow facility of experimental segments was not different than control. Specifically, overexpression of aquaporin-1 in the inner meshwork resulted in an average facility change of −2.0 ± 9.2 %, while overexpression of aquaporin-1 in the resistance-generating region changed outflow facility by −3.2 ± 11.2 %. Taken together, these results indicate that a transcellular pathway, mediated by aquaporin-1, does not contribute significantly to bulk outflow through the conventional aqueous outflow tract of human eyes. PMID:18657536

  13. A delayed, gonadotropin-dependent and growth-factor mediated activation of the ERK1/2 cascade negatively regulates aromatase expression in granulosa cells*

    PubMed Central

    Andric, Nebojsa; Ascoli, Mario

    2006-01-01

    Human CG and hFSH elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors hCG and hFSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6–9 h. Both, the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of PKA, the epidermal growth factor receptor (EGFR) kinase, metalloproteases and MEK. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C (PKC). Since the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins we tested the effects of the inhibitors mentioned on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSHR. A MEK inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by EGF-like growth factors and that the delayed effect is partially mediated by PKC and acts as a negative regulator of aromatase expression. PMID:16973759

  14. FcgammaRIIb expression on human germinal center B lymphocytes.

    PubMed

    Macardle, Peter J; Mardell, Carolyn; Bailey, Sheree; Wheatland, Loretta; Ho, Alice; Jessup, Claire; Roberton, Donal M; Zola, Heddy

    2002-12-01

    IgG antibody can specifically suppress the antibody response to antigen. This has been explained by the hypothesis that signaling through the B cell antigen receptor is negatively modulated by the co-ligation of immunoglobulin with the receptor for IgG, FcgammaRIIb. We hypothesized that inhibitory signaling through FcgammaRIIb would be counter-productive in germinal center cells undergoing selection by affinity maturation, since these cells are thought to receive a survival/proliferative signal by interacting with antigen displayed on follicular dendritic cells. We have identified and characterized a population of B lymphocytes with low/negative FcgammaRIIb expression that are present in human tonsil. Phenotypically these cells correspond to germinal center B cells and comprise both centroblast and centrocyte populations. In examining expression at the molecular level we determined that these B cells do not express detectable mRNA for FcgammaRIIb. We examined several culture conditions to induce expression of FcgammaRIIb on germinal center cells but could not determine conditions that altered expression. We then examined the functional consequence of cross-linking membrane immunoglobulin and the receptor for IgG on human B lymphocytes. Our results cast some doubt on the value of anti-IgG as a model for antigen-antibody complexes in studying human B cell regulation.

  15. Characterization of human septic sera induced gene expression modulation in human myocytes

    PubMed Central

    Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

    2009-01-01

    To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

  16. Immunodetection of Human LINE-1 Expression in Cultured Cells and Human Tissues.

    PubMed

    Sharma, Reema; Rodić, Nemanja; Burns, Kathleen H; Taylor, Martin S

    2016-01-01

    Long interspersed element-1 (LINE-1) is the only active protein-coding retrotransposon in humans. It is not expressed in somatic tissue but is aberrantly expressed in a wide variety of human cancers. ORF1p protein is the most robust indicator of LINE-1 expression; the protein accumulates in large quantities in cellular cytoplasm. Recently, monoclonal antibodies have allowed more complete characterizations of ORF1p expression and indicated potential for developing ORF1p as a clinical biomarker. Here, we describe a mouse monoclonal antibody specific for human LINE-1 ORF1p and its application in immunofluorescence and immunohistochemistry of both cells and human tissues. We also describe detection of tagged LINE-1 ORF2p via immunofluorescence. These general methods may be readily adapted to use with many other proteins and antibodies.

  17. Heterologous expression of human H1 histones in yeast.

    PubMed

    Albig, W; Runge, D M; Kratzmeier, M; Doenecke, D

    1998-09-18

    The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast. Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones. For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid. The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively. This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

  18. Distribution of cellular HSV-1 receptor expression in human brain.

    PubMed

    Lathe, Richard; Haas, Juergen G

    2016-12-15

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

  19. Cellular reprogramming of human amniotic fluid cells to express insulin.

    PubMed

    Gage, Blair K; Riedel, Michael J; Karanu, Francis; Rezania, Alireza; Fujita, Yukihiro; Webber, Travis D; Baker, Robert K; Wideman, Rhonda D; Kieffer, Timothy J

    2010-01-01

    Islet transplantation represents a potential cure for type 1 diabetes; however, a lack of sufficient donor material limits its clinical use. To address the shortfall of islet availability, surrogate insulin-producing cells are sought. Studies suggest that human amniotic fluid (hAF) contains multipotent progenitor cells capable of differentiating to all three germ layers. Here, we used high-content, live-cell imaging to assess the ability to reprogram hAF cells towards a beta cell phenotype. A fluorescent reporter system was developed where DsRed express (DSRE) expression is driven by the human insulin promoter. Using integrative lentiviral technology, we created stable reporter hAF cells that could be routinely monitored for insulin promoter activation. These cells were subjected to combinatorial high-content screening using adenoviral-mediated expression of up to six transcription factors important for beta cell development. Cells were monitored for DSRE expression which revealed an optimal combination of the transcription factors required to induce insulin gene expression in hAF cells. These optimally induced cells were examined for expression of additional beta cell transcription factors and proteins involved in glucose sensing and insulin processing. RT-qPCR revealed very low level expression of insulin that was ultimately insufficient to reverse streptozotocin-induced diabetes following sub-capsular kidney transplantation. High-content, live-cell imaging using fluorescent reporter cells provides a convenient method for repeated assessment of cellular reprogramming. hAF cells could be reprogrammed to express key beta cell proteins, however insulin gene expression was insufficient to reverse hyperglycemia in diabetic animals. Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  20. Serotoninergic and melatoninergic systems are fully expressed in human skin.

    PubMed

    Slominski, Andrzej; Pisarchik, Alexander; Semak, Igor; Sweatman, Trevor; Wortsman, Jacobo; Szczesniewski, Andre; Slugocki, George; McNulty, John; Kauser, Söbia; Tobin, Desmond J; Jing, Chen; Johansson, Olle

    2002-06-01

    We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N-acetyltransferase (AANAT), and hydroxyindole-O-methyltransferase (HIOMT). The products of AANAT activity were identified by RP-HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N-acetylserotonin (NAS) was detected by RP-HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.

  1. Expression of glucocorticoid receptors in the regenerating human skeletal muscle.

    PubMed

    Filipović, D; Pirkmajer, S; Mis, K; Mars, T; Grubic, Z

    2011-01-01

    Many stress conditions are accompanied by skeletal muscle dysfunction and regeneration, which is essentially a recapitulation of the embryonic development. However, regeneration usually occurs under conditions of hypothalamus-pituitary-adrenal gland axis activation and therefore increased glucocorticoid (GC) levels. Glucocorticoid receptor (GR), the main determinant of cellular responsiveness to GCs, exists in two isoforms (GRalpha and GRbeta) in humans. While the role of GRalpha is well characterized, GRbeta remains an elusive player in GC signalling. To elucidate basic characteristics of GC signalling in the regenerating human skeletal muscle we assessed GRalpha and GRbeta expression pattern in cultured human myoblasts and myotubes and their response to 24-hour dexamethasone (DEX) treatment. There was no difference in GRalpha mRNA and protein expression or DEX-mediated GRalpha down-regulation in myoblasts and myotubes. GRbeta mRNA level was very low in myoblasts and remained unaffected by differentiation and/or DEX. GRbeta protein could not be detected. These results indicate that response to GCs is established very early during human skeletal muscle regeneration and that it remains practically unchanged before innervation is established. Very low GRbeta mRNA expression and inability to detect GRbeta protein suggests that GRbeta is not a major player in the early stages of human skeletal muscle regeneration.

  2. [Generation of transgenic mice expressing human lysozyme in mammary gland].

    PubMed

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang

    2005-10-01

    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

  3. Anticancer drug bortezomib increases interleukin-8 expression in human monocytes.

    PubMed

    Sanacora, Shannon; Urdinez, Joaquin; Chang, Tzu-Pei; Vancurova, Ivana

    2015-05-01

    Bortezomib (BZ) is the first clinically approved proteasome inhibitor that has shown remarkable anticancer activity in patients with hematological malignancies. However, many patients relapse and develop resistance; yet, the molecular mechanisms of BZ resistance are not fully understood. We have recently shown that in solid tumors, BZ unexpectedly increases expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8), while it inhibits expression of other NFκB-regulated genes. Since monocytes and macrophages are major producers of IL-8, the goal of this study was to test the hypothesis that BZ increases the IL-8 expression in human monocytes and macrophages. Here, we show that BZ dramatically increases the IL-8 expression in lipopolysaccharide (LPS)-stimulated U937 macrophages as well as in unstimulated U937 monocytes and peripheral blood mononuclear cells, while it inhibits expression of IL-6, IL-1 and tumor necrosis factor-α. In addition, our results show that the underlying mechanisms involve p38 mitogen-activated protein kinase, which is required for the BZ-induced IL-8 expression. Together, these data suggest that the BZ-increased IL-8 expression in monocytes and macrophages may represent one of the mechanisms responsible for the BZ resistance and indicate that targeting the p38-mediated IL-8 expression could enhance the BZ effectiveness in cancer treatment.

  4. TP53 mutations, expression and interaction networks in human cancers

    PubMed Central

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-01

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers. PMID:27880943

  5. TP53 mutations, expression and interaction networks in human cancers.

    PubMed

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-03

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers.

  6. On Expression Patterns and Developmental Origin of Human Brain Regions.

    PubMed

    Kirsch, Lior; Chechik, Gal

    2016-08-01

    Anatomical substructures of the human brain have characteristic cell-types, connectivity and local circuitry, which are reflected in area-specific transcriptome signatures, but the principles governing area-specific transcription and their relation to brain development are still being studied. In adult rodents, areal transcriptome patterns agree with the embryonic origin of brain regions, but the processes and genes that preserve an embryonic signature in regional expression profiles were not quantified. Furthermore, it is not clear how embryonic-origin signatures of adult-brain expression interplay with changes in expression patterns during development. Here we first quantify which genes have regional expression-patterns related to the developmental origin of brain regions, using genome-wide mRNA expression from post-mortem adult human brains. We find that almost all human genes (92%) exhibit an expression pattern that agrees with developmental brain-region ontology, but that this agreement changes at multiple phases during development. Agreement is particularly strong in neuron-specific genes, but also in genes that are not spatially correlated with neuron-specific or glia-specific markers. Surprisingly, agreement is also stronger in early-evolved genes. We further find that pairs of similar genes having high agreement to developmental region ontology tend to be more strongly correlated or anti-correlated, and that the strength of spatial correlation changes more strongly in gene pairs with stronger embryonic signatures. These results suggest that transcription regulation of most genes in the adult human brain is spatially tuned in a way that changes through life, but in agreement with development-determined brain regions.

  7. On Expression Patterns and Developmental Origin of Human Brain Regions

    PubMed Central

    Kirsch, Lior; Chechik, Gal

    2016-01-01

    Anatomical substructures of the human brain have characteristic cell-types, connectivity and local circuitry, which are reflected in area-specific transcriptome signatures, but the principles governing area-specific transcription and their relation to brain development are still being studied. In adult rodents, areal transcriptome patterns agree with the embryonic origin of brain regions, but the processes and genes that preserve an embryonic signature in regional expression profiles were not quantified. Furthermore, it is not clear how embryonic-origin signatures of adult-brain expression interplay with changes in expression patterns during development. Here we first quantify which genes have regional expression-patterns related to the developmental origin of brain regions, using genome-wide mRNA expression from post-mortem adult human brains. We find that almost all human genes (92%) exhibit an expression pattern that agrees with developmental brain-region ontology, but that this agreement changes at multiple phases during development. Agreement is particularly strong in neuron-specific genes, but also in genes that are not spatially correlated with neuron-specific or glia-specific markers. Surprisingly, agreement is also stronger in early-evolved genes. We further find that pairs of similar genes having high agreement to developmental region ontology tend to be more strongly correlated or anti-correlated, and that the strength of spatial correlation changes more strongly in gene pairs with stronger embryonic signatures. These results suggest that transcription regulation of most genes in the adult human brain is spatially tuned in a way that changes through life, but in agreement with development-determined brain regions. PMID:27564987

  8. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  9. The effect of the intracervical administration of FSH or LH on the levels of hyaluronan, COX2, and COX2 mRNA in the cervix of the nonpregnant ewe.

    PubMed

    Leethongdee, Sukanya; Khalid, Muhammad; Scaramuzzi, Rex J

    2016-12-01

    During the periovulatory period, the cervix relaxes in response to changes in circulating concentrations of reproductive hormones. The present study investigated the role of gonadotrophins in cervical function by examining the expression of cyclooxygenase-2 (COX2) and COX2 mRNA and the concentration of hyaluronan (HA) in the cervix, after intracervical treatment with either FSH or LH. Eighteen ewes were assigned to four groups. They were then treated with commercial intravaginal progestagen sponges and eCG to synchronize their estrous cycles. Intracervical treatments were given 24 hours after removal of the sponges as follows: group 1: FSH, 2 mg; group 2: LH, 2 mg; group 3: vehicle; and group 4: control. Cervices were collected 54 hours after sponge removal and then divided into three regions. The expression of COX2 and COX2 mRNA was determined by immunohistochemistry and in situ hybridization and those of HA by ELISA. The levels of expression of COX2, COX2 mRNA, and HA were compared in six tissue layers (luminal epithelium, subepithelial stroma, circular, longitudinal and transverse muscle, and serosa) and in three cervical regions (vaginal, mid, and uterine). The results showed that both FSH and LH significantly increased the levels the COX2 mRNA and COX2 in the cervix, but the effects of the gonadotrophins were selective. The effects of both FSH and LH were most evident at the vaginal end of the cervix and least at the uterine end of the cervix. Furthermore, their effects were confined to the stroma and smooth muscle layers of the cervix in the case of FSH and to smooth muscle only in the case of LH. Neither FSH nor LH affected the concentration of HA in the cervix although FSH but not LH reduced the concentration of HA in cervical mucus. These findings suggest that the gonadotrophins regulate the expression of COX2 in the cervix and that they may have a role facilitating relaxation of the cervix during estrus in the ewe.

  10. Highly purified hMG versus recombinant FSH plus recombinant LH in intrauterine insemination cycles in women ≥35 years: a RCT.

    PubMed

    Moro, Francesca; Scarinci, Elisa; Palla, Carola; Romani, Federica; Familiari, Alessandra; Tropea, Anna; Leoncini, Emanuele; Lanzone, Antonio; Apa, Rosanna

    2015-01-01

    .3%) in the recombinant group versus 35/289 (12.2%) in the HP-hMG group [(odds ratio (OR) 1.50, 95% CI 0.94-2.41, P = 0.09]. The number of interrupted cycles for high risk of OHSS was 13/290 (4.5%) in the rFSH plus rLH group and 2/289 (0.7%) in the HP-hMG group (OR 6.73, 95% CI 1.51-30.12, P = 0.013). One of the limitations of this study was the early closure and the ongoing PR could be overestimated. Both patient and gynaecologist were informed of the assigned treatment. Our results demonstrated the lack of differences in terms of ongoing PR between recombinant product and HP-hMG, in women ≥35 years undergoing controlled ovarian stimulation for IUI cycles. HP-hMG was safer than recombinant gonadotrophin concerning the risk of OHSS. None. NCT01604044. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Quality of human milk expressed in a human milk bank and at home.

    PubMed

    Borges, Mayla S; Oliveira, Angela M de M; Hattori, Wallisen T; Abdallah, Vânia O S

    2017-08-30

    To evaluate the quality of the human milk expressed at home and at a human milk bank. This a retrospective, analytical, and observational study, performed by assessing titratable acidity records and the microbiological culture of 100 human milk samples expressed at home and at a human milk bank, in 2014. For the statistical analysis, generalized estimating equations (GEE) and the chi-squared test were used. When comparing the two sample groups, no significant difference was found, with 98% and 94% of the samples being approved among those collected at the milk bank and at home, respectively. No main interaction effect between local and titratable acidity records (p=0.285) was observed, and there was no statistically significant difference between the expected and observed values for the association between the collection place and the microbiological culture results (p=0.307). The quality of human milk expressed at home and at the milk bank are in agreement with the recommended standards, confirming that the expression of human milk at home is as safe as expression at the human milk bank, provided that the established hygiene, conservation, storage, and transport standards are followed. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  12. LIN28A expression reduces sickling of cultured human erythrocytes.

    PubMed

    de Vasconcellos, Jaira F; Fasano, Ross M; Lee, Y Terry; Kaushal, Megha; Byrnes, Colleen; Meier, Emily R; Anderson, Molly; Rabel, Antoinette; Braylan, Raul; Stroncek, David F; Miller, Jeffery L

    2014-01-01

    Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.

  13. Cystic fibrosis transmembrane conductance regulator expression in human hypothalamus.

    PubMed

    Mulberg, A E; Weyler, R T; Altschuler, S M; Hyde, T M

    1998-01-05

    We have previously characterized the expression of the cystic fibrosis transmembrane conductance regulator protein (CFTR) gene, mRNA and protein in rat brain with reverse transcriptase (RT)-PCR amplification, in situ hybridization and immunocytochemistry. We now report that the CFTR mRNA is expressed in the human anterior hypothalamus, an area involved in regulation of appetite, resting energy expenditure and sexual differentiation. Expression of CFTR in neurons localized to this region may elucidate the pathogenesis of other non-pulmonary manifestations of cystic fibrosis which commonly are observed in children with CF, including congenital absence of the vas deferens. Neuron-specific expression of CFTR in brain may be involved in the regulation of homeostatic functions including reproductive function and fertility through effects on neurosecretion, i.e. GnRH release. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain my lead to alteration in physiological function.

  14. An FSH and TSH pituitary adenoma, presenting with precocious puberty and central hyperthyroidism.

    PubMed

    Vargas, Guadalupe; Balcazar-Hernandez, Lourdes-Josefina; Melgar, Virgilio; Magriña-Mercado, Roser-Montserrat; Gonzalez, Baldomero; Baquera, Javier; Mercado, Moisés

    2017-01-01

    A 19-year-old woman with a history of isosexual precocious puberty and bilateral oophorectomy at age 10 years because of giant ovarian cysts, presents with headaches and mild symptoms and signs of hyperthyroidism. Hormonal evaluation revealed elevated FSH and LH levels in the postmenopausal range and free hyperthyroxinemia with an inappropriately normal TSH. Pituitary MRI showed a 2-cm macroadenoma with suprasellar extension. She underwent successful surgical resection of the pituitary tumor, which proved to be composed of two distinct populations of cells, each of them strongly immunoreactive for FSH and TSH, respectively. This mixed adenoma resulted in two different hormonal hypersecretion syndromes: the first one during childhood and consisting of central precocious puberty and ovarian hyperstimulation due to the excessive secretion of biologically active FSH and which was not investigated in detail and 10 years later, central hyperthyroidism due to inappropriate secretion of biologically active TSH. Although infrequent, two cases of isosexual central precocious puberty in girls due to biologically active FSH secreted by a pituitary adenoma have been previously reported in the literature. However, this is the first reported case of a mixed adenoma capable of secreting both, biologically active FSH and TSH. Although functioning gonadotrophinomas are infrequent, they should be included in the differential diagnosis of isosexual central precocious puberty.Some functioning gonadotrophinomas are mixed adenomas, secreting other biologically active hormones besides FSH, such as TSH.Early recognition and appropriate treatment of these tumors by transsphenoidal surgery is crucial in order to avoid unnecessary therapeutic interventions that may irreversibly compromise gonadal function.

  15. Superovulation of goats with purified pFSH supplemented with defined amounts of pLH.

    PubMed

    Nowshari, M A; Backers, J F; Holtz, W

    1995-03-01

    The superovulatory response of goats treated with purified pFSH supplemented with 30, 40 or 50% pLH was compared. Sixty-four Boer goat does were synchronized by progestagen-containing ear implant, randomly allotted to 3 groups and, beginning 2 d before implant removal, treated with purified pFSH supplemented with 30, 40 or 50% pLH. Each animal received 16 Armour Units of pFSH administered in 6 descending doses at 12-h intervals. Along with the last 2 injections, the does received 5 mg PGF(2alpha). Embryos were flushed either surgically or after slaughter on Day 5 or 6 after the last day of standing estrus. The percentage of animals responding to treatment was not different among groups treated with pFSH supplemented with 30, 40 or 50% pLH (76, 71 and 63%, respectively). The corresponding data for number of ovulations was 11.3 +/- 1.6, 16.3 +/- 1.8 and 16.4 +/- 2.6, for number of ova and embryos recovered 8.1 +/- 1.9, 12.0 +/- 1.5 and 13.5 +/- 2.9 and for number of transferable embryos 6.6 +/- 1.9, 9.1 +/- 1.5 and 7.1 +/- 2.1 (x +/- SEM). Results confirm the earlier finding of a good response of goats to pFSH preparations with a high FSH:LH ratio, and, although group differences were statistically nonsignificant (P > 0.05), they suggest that supplementation with approximately 40% pLH may be close to the optimum.

  16. An FSH and TSH pituitary adenoma, presenting with precocious puberty and central hyperthyroidism

    PubMed Central

    Vargas, Guadalupe; Balcazar-Hernandez, Lourdes-Josefina; Melgar, Virgilio; Magriña-Mercado, Roser-Montserrat; Gonzalez, Baldomero; Baquera, Javier

    2017-01-01

    A 19-year-old woman with a history of isosexual precocious puberty and bilateral oophorectomy at age 10 years because of giant ovarian cysts, presents with headaches and mild symptoms and signs of hyperthyroidism. Hormonal evaluation revealed elevated FSH and LH levels in the postmenopausal range and free hyperthyroxinemia with an inappropriately normal TSH. Pituitary MRI showed a 2-cm macroadenoma with suprasellar extension. She underwent successful surgical resection of the pituitary tumor, which proved to be composed of two distinct populations of cells, each of them strongly immunoreactive for FSH and TSH, respectively. This mixed adenoma resulted in two different hormonal hypersecretion syndromes: the first one during childhood and consisting of central precocious puberty and ovarian hyperstimulation due to the excessive secretion of biologically active FSH and which was not investigated in detail and 10 years later, central hyperthyroidism due to inappropriate secretion of biologically active TSH. Although infrequent, two cases of isosexual central precocious puberty in girls due to biologically active FSH secreted by a pituitary adenoma have been previously reported in the literature. However, this is the first reported case of a mixed adenoma capable of secreting both, biologically active FSH and TSH. Learning points: Although functioning gonadotrophinomas are infrequent, they should be included in the differential diagnosis of isosexual central precocious puberty. Some functioning gonadotrophinomas are mixed adenomas, secreting other biologically active hormones besides FSH, such as TSH. Early recognition and appropriate treatment of these tumors by transsphenoidal surgery is crucial in order to avoid unnecessary therapeutic interventions that may irreversibly compromise gonadal function. PMID:28721217

  17. KiSS-1 expression in human breast cancer.

    PubMed

    Martin, Tracey A; Watkins, Gareth; Jiang, Wen G

    2005-01-01

    The KiSS-1 gene encodes a 145 amino acid residue peptide that is further processed to a final peptide, metastin, a ligand to a G-coupled orphan receptor (OT7T175/AXOR12). KiSS-1 has been identified as a putative human metastasis suppressor gene in melanomas and in breast cancer cell lines. This study aimed to determine the expression and distribution of KiSS-1 and its receptor in human breast cancer tissues and to identify a possible link between expression levels and patient prognosis. Frozen sections from breast cancer primary tumours (matched tumour 124 and background 33) were immuno-stained with KiSS-1 antibody. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, and normalized with cytokeratin-19 levels). Levels of expression of KiSS-1 were higher in tumour compared to background tissues (3,124+/-1,262 vs 2,397+/-1,181) and significantly increased in node positive tumours compared to node negative (3,637+/-1,719 vs 2,653+/-1,994, P = 0.02). KiSS-1 expression was also increased with increasing grade and TNM status. There were no such trends with the KiSS-1 receptor. Expression of KiSS-1 was higher in patients who had died from breast cancer than those who had remained healthy (4,631+/-3,024 vs 2,280+/-1,403) whereas expression of the receptor was reduced (480+/-162 vs 195+/-134). Immunohistochemical staining showed increased expression of KiSS-1 in tumour sections. Insertion of the KiSS-1 gene into the human breast cancer cell line MDA-MB-231, resulted in cells that were significantly more motile and invasive in behaviour, with reduced adhesion to matrix, using respective assays. In conclusion, KiSS-1 expression is increased in human breast cancer, particularly in patients with aggressive tumours and with mortality. Over-expression of KiSS-1 in breast cancer cells result in more aggressive phenotype. Together, it suggests that KiSS-1 plays a role beyond the initial metastasis repressor in this cancer type.

  18. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  19. Exocyst complex protein expression in the human placenta.

    PubMed

    Gonzalez, I M; Ackerman, W E; Vandre, D D; Robinson, J M

    2014-07-01

    Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion. Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. D2-40/podoplanin expression in the human placenta.

    PubMed

    Wang, Y; Sun, J; Gu, Y; Zhao, S; Groome, L J; Alexander, J S

    2011-01-01

    Placental tissue expresses many lymphatic markers. The current study was undertaken to examine if D2-40/podoplanin, a lymphatic endothelial marker, was expressed in the human placenta, and how it is altered developmentally and pathologically. We examined D2-40/podoplanin and VEGFR-3 expressions in placentas from normotensive pregnancies at different gestational ages and in placentas from women with clinically defined preeclampsia. D2-40 expression in systemic lymphatic vessel endothelium served as a positive control. Protein expression for D2-40, VEGFR-3, and β-actin was determined by Western blot in placentas from normotensive (n = 6) and preeclamptic (n = 5) pregnancies. Our results show that D2-40/podoplanin was strongly expressed in the placenta, mainly as a network plexus pattern in the villous stroma throughout gestation. CD31 was limited to villous core fetal vessel endothelium and VEGFR-3 was found in both villous core fetal vessel endothelium and trophoblasts. D2-40/podoplanin expression was significantly decreased, and VEGFR-3 significantly increased in preeclamptic placental tissues compared to normotensive placental controls. Placental villous stroma is a reticular-like structure, and the localization of D2-40 to the stroma suggests that a lymphatic-like conductive network may exist in the human placenta. D2-40/podoplanin is an O-linked sialoglycoprotein. Although little is known regarding biological functions of sialylated glycoproteins within the placenta, placental D2-40/podoplanin may support fetal vessel angiogenesis during placenta development and reduced D2-40/podoplanin expression in preeclamptic placenta may contribute to altered interstitial fluid homeostasis and impaired angiogenesis in this pregnancy disorder. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Neural decoding of expressive human movement from scalp electroencephalography (EEG)

    PubMed Central

    Cruz-Garza, Jesus G.; Hernandez, Zachery R.; Nepaul, Sargoon; Bradley, Karen K.; Contreras-Vidal, Jose L.

    2014-01-01

    Although efforts to characterize human movement through electroencephalography (EEG) have revealed neural activities unique to limb control that can be used to infer movement kinematics, it is still unknown the extent to which EEG can be used to discern the expressive qualities that influence such movements. In this study we used EEG and inertial sensors to record brain activity and movement of five skilled and certified Laban Movement Analysis (LMA) dancers. Each dancer performed whole body movements of three Action types: movements devoid of expressive qualities (“Neutral”), non-expressive movements while thinking about specific expressive qualities (“Think”), and enacted expressive movements (“Do”). The expressive movement qualities that were used in the “Think” and “Do” actions consisted of a sequence of eight Laban Effort qualities as defined by LMA—a notation system and language for describing, visualizing, interpreting and documenting all varieties of human movement. We used delta band (0.2–4 Hz) EEG as input to a machine learning algorithm that computed locality-preserving Fisher's discriminant analysis (LFDA) for dimensionality reduction followed by Gaussian mixture models (GMMs) to decode the type of Action. We also trained our LFDA-GMM models to classify all the possible combinations of Action Type and Laban Effort quality (giving a total of 17 classes). Classification accuracy rates were 59.4 ± 0.6% for Action Type and 88.2 ± 0.7% for Laban Effort quality Type. Ancillary analyses of the potential relations between the EEG and movement kinematics of the dancer's body, indicated that motion-related artifacts did not significantly influence our classification results. In summary, this research demonstrates that EEG has valuable information about the expressive qualities of movement. These results may have applications for advancing the understanding of the neural basis of expressive movements and for the development of

  2. Neural decoding of expressive human movement from scalp electroencephalography (EEG).

    PubMed

    Cruz-Garza, Jesus G; Hernandez, Zachery R; Nepaul, Sargoon; Bradley, Karen K; Contreras-Vidal, Jose L

    2014-01-01

    Although efforts to characterize human movement through electroencephalography (EEG) have revealed neural activities unique to limb control that can be used to infer movement kinematics, it is still unknown the extent to which EEG can be used to discern the expressive qualities that influence such movements. In this study we used EEG and inertial sensors to record brain activity and movement of five skilled and certified Laban Movement Analysis (LMA) dancers. Each dancer performed whole body movements of three Action types: movements devoid of expressive qualities ("Neutral"), non-expressive movements while thinking about specific expressive qualities ("Think"), and enacted expressive movements ("Do"). The expressive movement qualities that were used in the "Think" and "Do" actions consisted of a sequence of eight Laban Effort qualities as defined by LMA-a notation system and language for describing, visualizing, interpreting and documenting all varieties of human movement. We used delta band (0.2-4 Hz) EEG as input to a machine learning algorithm that computed locality-preserving Fisher's discriminant analysis (LFDA) for dimensionality reduction followed by Gaussian mixture models (GMMs) to decode the type of Action. We also trained our LFDA-GMM models to classify all the possible combinations of Action Type and Laban Effort quality (giving a total of 17 classes). Classification accuracy rates were 59.4 ± 0.6% for Action Type and 88.2 ± 0.7% for Laban Effort quality Type. Ancillary analyses of the potential relations between the EEG and movement kinematics of the dancer's body, indicated that motion-related artifacts did not significantly influence our classification results. In summary, this research demonstrates that EEG has valuable information about the expressive qualities of movement. These results may have applications for advancing the understanding of the neural basis of expressive movements and for the development of neuroprosthetics to restore

  3. Expression of testicular androgen receptor in non-obstructive azoospermia and its change after hormonal therapy.

    PubMed

    Kato, Y; Shiraishi, K; Matsuyama, H

    2014-09-01

    Several trials aimed at improving the sperm retrieval from men with non-obstructive azoospermia (NOA) by optimizing intratesticular testosterone (ITT) have reported partial responses, however, an appropriate level of ITT has not been identified. In this study, we examined the expression of the testicular androgen receptor (AR) in NOA and investigated its correlation with clinical and pathological parameters. Expression of the testicular AR was investigated in 52 men with NOA and 22 men with obstructive azoospermia (OA). Twenty-two patients for whom sperm retrieval failed during microdissection testicular sperm extraction (micro-TESE) were enrolled in hormonal therapy using hCG with or without recombinant human follicle stimulating hormone (rhFSH) prior to a second micro-TESE. Sertoli cells were identified by vimentin immunostaining, and positivity in Sertoli cells was used as the AR index. AR immunostaining was robust in the nuclei of Sertoli cells [Sertoli cell androgen receptor (SCAR)] in both OA and NOA. The mean AR index in NOA was significantly higher than that in OA (p < 0.05). In NOA patients, there was no correlation between the AR index and the clinical parameters, whereas the AR index of early maturation arrest (MA) was significantly lower than that of Sertoli cell only, late MA and hypospermatogenesis (p < 0.05). A significant increase in the AR index after salvage hormonal therapy was shown, particularly when using rhFSH. The AR index in patients from whom spermatozoa could be retrieved at the second micro-TESE increased significantly after hormonal therapy. In human testes, the expression of AR is dominant in Sertoli cells, and the expression of SCAR is upregulated by FSH. Germ cell maturation, especially during spermatogonia to spermatocyte stage, has been shown to be SCAR-dependent. Taken together, the results indicate that SCAR elevation is closely associated with sperm retrieval after hormonal therapy and that FSH-based hormonal therapy is

  4. Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

    PubMed

    Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

    2017-03-01

    To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human

  5. Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells.

    PubMed

    Zhou, Yang; Ren, Linzhu; Zhu, Jianguo; Yan, Sen; Wang, Haijun; Song, Na; Li, Li; Ouyang, Hongsheng; Pang, Daxin

    2011-08-01

    Human Fibroblast growth factor 1 (FGF1) has been recognized as a valuable protein drug for the treatment of many diseases because of its multiple functions in regulating a variety of biological processes involved in embryonic development, cell growth and differentiation, morphogenesis, tissue repair, and others. The aim of this study was to develop an FGF1 mammary gland-specific expression vector to produce FGF1 on a large scale from transgenic cows to meet the demand for FGF1 in medical use. In this study, we generated an FGF1 mammary gland-specific expression vector and validated its function in human MCF-7 cells. This vector was shown to successfully express functional FGF1, thus potentially enabling the generation of transgenic cows to be used as mammary gland bioreactors.

  6. Heterogeneous expression of apolipoprotein-E by human macrophages

    PubMed Central

    Tedla, Nicodemus; Glaros, Elias N; Brunk, Ulf T; Jessup, Wendy; Garner, Brett

    2004-01-01

    Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3–5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression. PMID:15500620

  7. Expression and modulation of CD44 variant isoforms in humans

    PubMed Central

    1994-01-01

    CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines. PMID:7507492

  8. Successful use of aromatase inhibitor letrozole in NOA with an elevated FSH level: a case report.

    PubMed

    Zhao, D; Pan, L; Zhang, F; Pan, F; Ma, J; Zhang, X; Liu, Y

    2014-05-01

    Aromatase inhibitors inhibit the conversion of testosterone to oestrogens and could reduce serum oestradiol concentrations. Letrozole is one of aromatase inhibitors frequently used in treatment of men with oligospermia. We present the case of an infertile man with small testes and an elevated FSH level, which was diagnosed as NOA, hypospermatogenesis proven by testicular biopsy. After taking letrozole for 3 months, semen analyses by computer-aided sperm analysis present that this man had normal spermatogenesis. This is the first case report of the activation of spermatogenesis, in man who was NOA with elevated FSH level, resulting from the use of the one of aromatase inhibitors.

  9. Prognostic significance of PLIN1 expression in human breast cancer

    PubMed Central

    Zhou, Cefan; Wang, Ming; Zhou, Li; Zhang, Yi; Liu, Weiyong; Qin, Wenying; He, Rong; Lu, Yang; Wang, Yefu; Chen, Xing-Zhen; Tang, Jingfeng

    2016-01-01

    Breast cancer is a heterogeneous disease associated with diverse clinical, biological and molecular features, presenting huge challenges for prognosis and treatment. Here we found that perilipin-1 (PLIN1) mRNA expression is significantly downregulated in human breast cancer. Kaplan-Meier analysis indicated that patients presenting with reduced PLIN1 expression exhibited poorer overall metastatic relapse-free survival (p = 0.03). Further Cox proportional hazard models analysis revealed that the reduced expression of PLIN1 is an independent predictor of overall survival in estrogen receptor positive (p < 0.0001, HR = 0.87, 95% CI = 0.81–0.92, N = 3,600) and luminal A-subtype (p = 0.02, HR = 0.88, 95% CI = 0.78–0.98, N = 1,469) breast cancer patients. We also demonstrated that the exogenous expression of PLIN1 in human breast cancer MCF-7 and MDA-MB-231 cells significantly inhibits cell proliferation, migration, invasion and in vivo tumorigenesis in mice. Together, these data provide novel insights into a prognostic significance of PLIN1 in human breast cancer and reveal a potentially new gene therapy target for breast cancer. PMID:27359054

  10. Aldosterone does not modify gene expression in human endothelial cells.

    PubMed

    Verhovez, A; Williams, T A; Morello, F; Monticone, S; Brizzi, M F; Dentelli, P; Fallo, F; Fabris, B; Amenta, F; Gomez-Sanchez, C; Veglio, F; Mulatero, P

    2012-03-01

    The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

  11. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    PubMed

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  12. Expression of Matrix Metalloproteinases in Human Breast Cancer Tissues

    PubMed Central

    Benson, Chellakkan Selvanesan; Babu, Somasundaram Dinesh; Radhakrishna, Selvi; Selvamurugan, Nagarajan; Sankar, Bhaskaran Ravi

    2013-01-01

    BACKGROUND: Breast cancer is the most common cancer affecting women in the world today. Matrix metalloproteinases (MMPs) are a family of endopeptidases that can degrade extracellular matrix proteins and promote cell invasion and metastasis. MMPs are differentially expressed and their expressions are often associated with a poor prognosis for patients. OBJECTIVE: The aim of this study is to investigate and compare the expression of MMPs in different grades of human breast cancer tissues with normal breast tissues. PATIENTS AND METHODS: We collected 39 breast cancer samples (24 grade II and 15 grade III) along with 16 normal breast tissues from outside the tumor margin during cancer removal surgery. The samples were analysed for the expression of all known MMPs using real-time quantitative PCR. RESULTS: The results indicate that mRNA expressions of MMP-1, -9,-11,-15,-24 and -25 were upregulated in breast cancer tissues when compared to normal breast tissues. But, the mRNA expressions of MMP-10 and MMP-19 were downregulated in cancer tissue. In membrane associated MMPs like MMP-15 and MMP-24 we found a grade dependent increase of their mRNA expression. CONCLUSION: Our studies demonstrate that MMPs are differentially regulated in breast cancer tissues and they might play various roles in tumor invasion, metastasis and angiogenesis. Thus, MMPs are of immense value to be studied as diagnostic markers and drug target. PMID:23568046

  13. Altered gene expression in human placenta after suspected preterm labour.

    PubMed

    Oros, D; Strunk, M; Breton, P; Paules, C; Benito, R; Moreno, E; Garcés, M; Godino, J; Schoorlemmer, J

    2017-07-01

    Suspected preterm labour occurs in around 9% of pregnancies. However, almost two-thirds of women admitted for threatened preterm labour ultimately deliver at term and are considered risk-free for fetal development. We examined placental and umbilical cord blood samples from preterm or term deliveries after threatened preterm labour as well as term deliveries without threatened preterm labour. We quantitatively analysed the mRNA expression of inflammatory markers (IL6, IFNγ, and TNFα) and modulators of angiogenesis (FGF2, PGF, VEGFA, VEGFB, and VEGFR1). A total of 132 deliveries were analysed. Preterm delivery and term delivery after suspected preterm labour groups showed similar increases in TNFα expression compared with the term delivery control group in umbilical cord blood samples. Placental samples from preterm and term deliveries after suspected preterm labour exhibited significantly increased expression of TNFα and IL6 and decreased expression of IFNγ. Suspected preterm labour was also associated with altered expression of angiogenic factors, although not all differences reached statistical significance. We found gene expression patterns indicative of inflammation in human placentas after suspected preterm labour regardless of whether the deliveries occurred preterm or at term. Similarly, a trend towards altered expression of angiogeneic factors was not limited to preterm birth. These findings suggest that the biological mechanisms underlying threatened preterm labour affect pregnancies independently of gestational age at birth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Esophageal human β-defensin expression in eosinophilic esophagitis.

    PubMed

    Schroeder, Shauna; Robinson, Zachary D; Masterson, Joanne C; Hosford, Lindsay; Moore, Wendy; Pan, Zhaoxing; Harris, Rachel; Souza, Rhonda F; Spechler, Stuart Jon; Fillon, Sophie A; Furuta, Glenn T

    2013-05-01

    Defensins are antimicrobial peptides expressed on mucosal surfaces that contribute to maintaining intestinal homeostasis by providing innate defense mechanisms for the epithelia. Defensin expression is altered in a number of diseases that affect mucosal surfaces, such as atopic dermatitis, allergic rhinitis, and inflammatory bowel disease. Similar to atopic dermatitis, eosinophilic esophagitis (EoE) is a chronic disease in which the squamous epithelial surface is affected by a similar TH2 microenvironment and eosinophil-predominant inflammation. Therefore, we hypothesized that defensin expression would be decreased in EoE. To address this, we measured defensin expression in vitro in cell lines derived from patients with EoE (EoE1-T) or gastroesophageal reflux disease (GERD) (NES-G4T cells) and ex vivo in esophageal mucosal biopsy samples from children with EoE or GERD and control children without esophageal disease. Interleukin-5 induced a decrease in human β-defensin (hBD) -1 and hBD3 expression in EoE1-T but not in NES-G4T cells. Compared with esophageal biopsy specimens from GERD and control children, specimens from EoE pediatric patients revealed a significant decrease in mRNA and protein expression for hBD1 and hBD3. Diminished expression of hBD1 and hBD3 may make the esophageal epithelium more susceptible to the development and/or perpetuation of EoE.

  15. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  16. Validation of a noninvasive diagnostic tool to verify neuter status in dogs: The urinary FSH to creatinine ratio.

    PubMed

    Albers-Wolthers, C H J; de Gier, J; Oei, C H Y; Schaefers-Okkens, A C; Kooistra, H S

    2016-09-15

    Determining the presence of functional gonadal tissue in dogs can be challenging, especially in bitches during anestrus or not known to have been ovariectomized, or in male dogs with nonscrotal testes. Furthermore, in male dogs treated with deslorelin, a slow-release GnRH agonist implant for reversible chemical castration, the verification of complete downregulation of the hypothalamic-pituitary-gonadal (HPG) axis can be difficult, especially if pretreatment parameters such as the size of the testes or prostate gland are not available. The aims of this study were to validate an immunoradiometric assay for measurement of FSH in canine urine, to determine if the urinary FSH to creatinine ratio can be used to verify the neuter status in bitches and male dogs, as an alternative to the plasma FSH concentration, and to determine if downregulation of the HPG axis is achieved in male dogs during deslorelin treatment. Recovery of added canine FSH and serial dilutions of urine reported that the immunoradiometric assay measures urinary FSH concentration accurately and with high precision. Plasma FSH concentrations (the mean of two samples, taken 40 minutes apart) and the urinary FSH to creatinine ratio were determined before gonadectomy and 140 days (median, range 121-225 days) and 206 days (median, range 158-294 days) after gonadectomy of 13 bitches and five male dogs, respectively, and in 13 male dogs before and 132 days (median, range 117-174 days) after administration of a deslorelin implant. In both bitches and male dogs, the plasma FSH concentration and the urinary FSH to creatinine ratio were significantly higher after gonadectomy, with no overlapping of their ranges. Receiver operating characteristic analysis of the urinary FSH to creatinine ratio revealed a cut-off value of 2.9 in bitches and 6.5 in males to verify the presence or absence of functional gonadal tissue. In male dogs treated with deslorelin, the plasma FSH concentrations and urinary FSH to

  17. Decoding NADPH oxidase 4 expression in human tumors.

    PubMed

    Meitzler, Jennifer L; Makhlouf, Hala R; Antony, Smitha; Wu, Yongzhong; Butcher, Donna; Jiang, Guojian; Juhasz, Agnes; Lu, Jiamo; Dahan, Iris; Jansen-Dürr, Pidder; Pircher, Haymo; Shah, Ajay M; Roy, Krishnendu; Doroshow, James H

    2017-10-01

    NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage. Published by Elsevier B.V.

  18. L1 expression and regulation in humans and rodents

    PubMed Central

    Rosser, James M.; An, Wenfeng

    2015-01-01

    Long interspersed elements type 1 (LINE-1s, or L1s) have impacted mammalian genomes at multiple levels. L1 transcription is mainly controlled by its 5’ untranslated region (5’UTR), which differs significantly among active human and rodent L1 families. In this review, L1 expression and its regulation are examined in the context of human and rodent development. First, endogenous L1 expression patterns in three different species—human, rat, and mouse—are compared and contrasted. A detailed account of relevant experimental evidence is presented according to the source material, such as cell lines, tumors, and normal somatic and germline tissues from different developmental stages. Second, factors involved in the regulation of L1 expression at both transcriptional and posttranscriptional levels are discussed. These include transcription factors, DNA methylation, PIWI-interacting RNAs (piRNAs), RNA interference (RNAi), and posttranscriptional host factors. Similarities and differences between human and rodent L1s are highlighted. Third, recent findings from transgenic mouse models of L1 are summarized and contrasted with those from endogenous L1 studies. Finally, the challenges and opportunities for L1 mouse models are discussed. PMID:22202032

  19. Human airway epithelia express catalytically active NEU3 sialidase.

    PubMed

    Lillehoj, Erik P; Hyun, Sang Won; Feng, Chiguang; Zhang, Lei; Liu, Anguo; Guang, Wei; Nguyen, Chinh; Sun, Wenji; Luzina, Irina G; Webb, Tonya J; Atamas, Sergei P; Passaniti, Antonino; Twaddell, William S; Puché, Adam C; Wang, Lai-Xi; Cross, Alan S; Goldblum, Simeon E

    2014-05-01

    Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

  20. Hierarchical Recognition Scheme for Human Facial Expression Recognition Systems

    PubMed Central

    Siddiqi, Muhammad Hameed; Lee, Sungyoung; Lee, Young-Koo; Khan, Adil Mehmood; Truc, Phan Tran Ho

    2013-01-01

    Over the last decade, human facial expressions recognition (FER) has emerged as an important research area. Several factors make FER a challenging research problem. These include varying light conditions in training and test images; need for automatic and accurate face detection before feature extraction; and high similarity among different expressions that makes it difficult to distinguish these expressions with a high accuracy. This work implements a hierarchical linear discriminant analysis-based facial expressions recognition (HL-FER) system to tackle these problems. Unlike the previous systems, the HL-FER uses a pre-processing step to eliminate light effects, incorporates a new automatic face detection scheme, employs methods to extract both global and local features, and utilizes a HL-FER to overcome the problem of high similarity among different expressions. Unlike most of the previous works that were evaluated using a single dataset, the performance of the HL-FER is assessed using three publicly available datasets under three different experimental settings: n-fold cross validation based on subjects for each dataset separately; n-fold cross validation rule based on datasets; and, finally, a last set of experiments to assess the effectiveness of each module of the HL-FER separately. Weighted average recognition accuracy of 98.7% across three different datasets, using three classifiers, indicates the success of employing the HL-FER for human FER. PMID:24316568

  1. Expression of the Endocannabinoid Receptors in Human Fascial Tissue

    PubMed Central

    Fede, C.; Albertin, G.; Petrelli, L.; Sfriso, M.M.; Biz, C.; Caro, R. De; Stecco, C.

    2016-01-01

    Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation. PMID:27349320

  2. Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression.

    PubMed

    Buldreghini, E; Hamada, A; Macrì, M L; Amoroso, S; Boscaro, M; Lenzi, A; Agarwal, A; Balercia, G

    2014-12-01

    In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46 ± 0.38 versus 7.06 ± 0.56, P = 0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

  3. Expression of aquaporin 1 (AQP1) in human synovitis.

    PubMed

    Mobasheri, Ali; Moskaluk, Christopher A; Marples, David; Shakibaei, Mehdi

    2010-04-20

    Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial proliferation (synovitis), articular cartilage and subchondral bone degradation as well as joint swelling. Joint swelling and edema often accompany pannus formation and chronic joint inflammation in RA. We have recently shown that human chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels and that AQP1 is upregulated in RA cartilage. Clinical evidence suggests that joint swelling and edema accompany the chronic inflammation observed in synovial joints of RA patients. Therefore we hypothesized that AQP1 water channels may be involved in joint swelling and synovial edema formation. To test this hypothesis, we performed immunostaining of normal and human synovitis tissue microarrays (TMAs) to investigate whether the expression of AQP1 water channels is altered in the synovium in synovitis. Immunohistochemistry revealed that AQP1 is expressed in synovial micro-vessels and synoviocytes from normal joints (n=20 normal subjects). Semi-quantitative histomorphometric analysis of AQP1 expression in the TMAs revealed upregulation of the membrane protein in the synovium derived from RA (n=10) and psoriatic arthritis (n=8) patients. These results indicate a potential role for synovial AQP1 and other aquaporins in joint swelling and the vasogenic edema fluid formation and hydrarthrosis associated with synovial inflammation. Future experiments will need to determine whether the expression of other aquaporins is altered in synovitis. Copyright 2010 Elsevier GmbH. All rights reserved.

  4. Identifying gene expression modules that define human cell fates.

    PubMed

    Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E

    2016-05-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Comparison of the Gene Expression Profiles of Human Hematopoietic Stem Cells between Humans and a Humanized Xenograft Model.

    PubMed

    Matsuzawa, Hideyuki; Matsushita, Hiromichi; Yahata, Takashi; Tanaka, Masayuki; Ando, Kiyoshi

    2017-04-20

    The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγ(null)(NOG) mice transplanted with human CD34(+)/CD38(-)/Lin(-/low) hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34(+)/CD38(-)/Lin(-/low) cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34(+)/CD38(-)/Lin(-/low) HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34(+)/CD38(-)/Lin(-/low) cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34(+)/CD38(-)/Lin(-/low) cells in BM. Our findings demonstrate that the xenograft model of human CB CD34(+)/CD38(-)/Lin(-/low) cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.

  6. Efficacy of a single intramuscular injection of porcine FSH in hyaluronan prior to ovum pick-up in Holstein cattle.

    PubMed

    Vieira, L M; Rodrigues, C A; Netto, A Castro; Guerreiro, B M; Silveira, C R A; Freitas, B G; Bragança, L G M; Marques, K N G; Sá Filho, M F; Bó, G A; Mapletoft, R J; Baruselli, P S

    2016-03-15

    Plasma FSH profiles, in vitro embryo production (IVP) after ovum pickup (OPU), and establishment of pregnancy with IVP embryos were compared in untreated Holstein oocyte donors and those superstimulated with multiple injections or a single intramuscular (IM) injection of porcine FSH (pFSH) in hyaluronan (HA). Plasma FSH profiles were determined in 23 heifers randomly allocated to one of four groups. Controls received no treatment, whereas the F200 group received 200 mg of pFSH in four doses, 12 hours apart. The F200HA and F300HA groups received 200- or 300-mg pFSH in 5 mL or 7.5 mL, respectively of a 0.5% HA solution by a single IM injection. Plasma FSH levels were determined before the first pFSH treatment and every 6 hours over 96 hours. All data were analyzed by orthogonal contrasts. Circulating FSH area under curve (AUC) in pFSH-treated animals was greater than that in the control group (P = 0.02). Although the AUC did not differ among FSH-treated groups (P = 0.56), the total period with elevated plasma FSH was greater in the F200 group than in the HA groups (P < 0.0001). However, the F300HA group had a greater AUC than the F200HA group (P = 0.006), with a similar total period with elevated plasma FSH (P = 0.17). The IVP was performed in 90 nonlactating Holstein cows randomly allocated to one of the four treatment groups as in the first experiment. A greater proportion of medium-sized (6-10 mm) follicles was observed in cows receiving pFSH, regardless of the treatment group (P < 0.0001). Also, numbers of follicles (P = 0.01), cumulus-oocyte complexes (COCs) retrieved (P = 0.01) and matured (P = 0.02), cleavage rates (P = 0.002), and blastocysts produced per OPU session (P = 0.06) were greater in cows receiving pFSH, regardless of the treatment group. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COCs cultured (P = 0.04), and blastocysts produced per OPU session (P = 0.06) than cows in the F300HA group. Similar pregnancy rates were

  7. Expression of Thy-1 on human hematopoietic progenitor cells

    PubMed Central

    1993-01-01

    Expression of Thy-1 on hematopoietic cells from human fetal liver (FL), cord blood (CB), and bone marrow (BM) was studied with a novel anti-Thy- 1 antibody, 5E10. Specificity of 5E10 for human Thy-1 was demonstrated by immunoprecipitation of a 25-35-kD molecule, and the sequence of a cDNA that was cloned by immunoselection of COS cells transfected with a cDNA library derived from a 5E10+ cell line. Two- and three-color immunofluorescence staining experiments revealed that the Thy-1 expression is restricted to, an average, 1-4% of FL, CB, and BM cells, and binding to these cell types is essentially restricted to a very small subset of lymphoid cells and approximately 25% of CD34+ cells. Thy-1+ CD34+ cells were further characterized as CD38lo/CD45RO+/CD45RA- /CD71lo/c-kit(lo) and rhodamine 123dull. When CD34+ cells were sorted on the basis of Thy-1 expression, the majority of clonogenic cells were recovered in the CD34+Thy-1- fraction, whereas the majority of cells capable of producing myeloid colonies after 5-8 wk of long-term culture (long-term culture initiating cells) were recovered in the Thy-1+CD34+ fraction. In addition to CD34+ cells, Thy-1 was found to be expressed on a variable, very small number (< 1%) of CD34- mononuclear cells in BM, CB, and peripheral blood that were further characterized as CD3+ CD4+ lymphocytes. The restricted expression of Thy-1 on primitive hematopoietic cells is in agreement with a previous report (Baum et al., 1992. Proc. Natl. Acad. Sci. USA. 89:2804) in which Thy-1 expression was used to enrich for primitive hematopoietic cells from fetal tissue. Compared with those previous studies, we found Thy-1 expression on a larger proportion of CD34+ cells (25% in our study vs. 5% in Baum et al.) and furthermore performed studies on Thy-1 expression on CD34+ cells from CB, FL, and BM in relation to markers that are known to be differentially expressed on hematopoietic cells. Taken together our results indicate that Thy-1-specific antibody

  8. Testis-specific expression of the human MYCL2 gene.

    PubMed Central

    Robertson, N G; Pomponio, R J; Mutter, G L; Morton, C C

    1991-01-01

    We have characterized the expression of MYCL2, an intronless X-linked gene related to MYCL1. RNase protection analysis of a panel of human normal and tumor tissues has revealed that MYCL2 is expressed almost exclusively in human adult normal testis; much lower levels of transcript were detected in one human lung adenocarcinoma. No MYCL2 transcript was found in human testis RNA obtained from second trimester fetuses. This observation suggests a germ cell rather than somatic cell origin of the transcript and possible developmental regulation of MYCL2. Northern blot analysis of poly(A)+ RNA from adult human normal testis with an antisense riboprobe revealed a transcript of approximately 4.8-kb, which is in agreement with the size predicted from the MYCL2 nucleotide sequence. Antisense transcripts were found spanning regions of MYCL2 corresponding to all three exons of MYCL1. No sizable open reading frame was seen for the MYCL2 antisense transcripts suggesting that they may represent either regulatory sequences or an intron of a gene encoded by the complementary strand. RNase protection assays and the 5' RACE protocol (Rapid Amplification of cDNA Ends) were used to address the localization of the transcription start site of the MYCL2 sense transcript and different putative promoters and transcription regulatory elements have been identified. Images PMID:1711681

  9. Agouti expression in human adipose tissue: functional consequences and increased expression in type 2 diabetes.

    PubMed

    Smith, Steven R; Gawronska-Kozak, Barbara; Janderová, Lenka; Nguyen, Taylor; Murrell, Angela; Stephens, Jacqueline M; Mynatt, Randall L

    2003-12-01

    It is well recognized that the agouti/melanocortin system is an important regulator of body weight homeostasis. Given that agouti is expressed in human adipose tissue and that the ectopic expression of agouti in adipose tissue results in moderately obese mice, the link between agouti expression in human adipose tissue and obesity/type 2 diabetes was investigated. Although there was no apparent relationship between agouti mRNA levels and BMI, agouti mRNA levels were significantly elevated in subjects with type 2 diabetes. The regulation of agouti in cultured human adipocytes revealed that insulin did not regulate agouti mRNA, whereas dexamethasone treatment potently increased the levels of agouti mRNA. Experiments with cultured human preadipocytes and with cells obtained from transgenic mice that overexpress agouti demonstrated that melanocortin receptor (MCR) signaling in adipose tissue can regulate both preadipocyte proliferation and differentiation. Taken together, these results reveal that agouti can regulate adipogenesis at several levels and suggest that there are functional consequences of elevated agouti levels in human adipose tissue. The influence of MCR signaling on adipogenesis combined with the well-established role of MCR signaling in the hypothalamus suggest that adipogenesis is coordinately regulated with food intake and energy expenditure.

  10. Human mature adipocytes express albumin and this expression is not regulated by inflammation.

    PubMed

    Sirico, Maria Luisa; Guida, Bruna; Procino, Alfredo; Pota, Andrea; Sodo, Maurizio; Grandaliano, Giuseppe; Simone, Simona; Pertosa, Giovanni; Riccio, Eleonora; Memoli, Bruno

    2012-01-01

    Our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. Both inflamed and healthy subjects provided adipose tissue samples. RT-PCR, Real-Time PCR, and Western Blot analysis on homogenates of adipocytes and pre-adipocytes were performed. In sixty-three healthy subjects and fifty-four micro-inflamed end stage renal disease (ESRD) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. A clear gene expression of albumin was showed in pre-adipocytes and, for the first time, in mature adipocytes. Albumin gene expression resulted significantly higher in pre-adipocytes than in adipocytes. No significant difference in albumin gene expression was showed between healthy controls and inflamed patients. A significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed ESRD patients. In the present study we found first time evidence that human adipocytes express albumin. Our results also showed that systemic inflammation does not modulate albumin gene expression. The negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin.

  11. Human Mature Adipocytes Express Albumin and This Expression Is Not Regulated by Inflammation

    PubMed Central

    Sirico, Maria Luisa; Guida, Bruna; Procino, Alfredo; Pota, Andrea; Sodo, Maurizio; Grandaliano, Giuseppe; Simone, Simona; Pertosa, Giovanni; Riccio, Eleonora; Memoli, Bruno

    2012-01-01

    Aims. Our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. Methods. Both inflamed and healthy subjects provided adipose tissue samples. RT-PCR, Real-Time PCR, and Western Blot analysis on homogenates of adipocytes and pre-adipocytes were performed. In sixty-three healthy subjects and fifty-four micro-inflamed end stage renal disease (ESRD) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. Results. A clear gene expression of albumin was showed in pre-adipocytes and, for the first time, in mature adipocytes. Albumin gene expression resulted significantly higher in pre-adipocytes than in adipocytes. No significant difference in albumin gene expression was showed between healthy controls and inflamed patients. A significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed ESRD patients. Conclusions. In the present study we found first time evidence that human adipocytes express albumin. Our results also showed that systemic inflammation does not modulate albumin gene expression. The negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin. PMID:22675238

  12. Polymorphic GGC repeat differentially regulates human reelin gene expression levels.

    PubMed

    Persico, A M; Levitt, P; Pimenta, A F

    2006-10-01

    The human gene encoding Reelin (RELN), a pivotal protein in neurodevelopment, includes a polymorphic GGC repeat in its 5' untranslated region (UTR). CHO cells transfected with constructs encompassing the RELN 5'UTR with 4-to-13 GGC repeats upstream of the luciferase reporter gene show declining luciferase activity with increasing GGC repeat number (P < 0.005), as predicted by computer-based simulations. Conversely, RELN 5'UTR sequences boost reporter gene expression above control levels in neuronal SN56 and N2A cell lines, but 12- and 13-repeat alleles still yield 50-60% less luciferase activity compared to the more common 8- and 10-repeat alleles (P < 0.0001). RELN "long" GGC alleles significantly blunt gene expression and may, through this effect, confer vulnerability to human disorders, such as schizophrenia and autism.

  13. Cadherin-11 expression is upregulated in invasive human breast cancer

    PubMed Central

    Pohlodek, Kamil; Tan, Yen Y.; Singer, Christian F.; Gschwantler-Kaulich, Daphne

    2016-01-01

    Loss of expression of cadherin-11 protein is correlated with a loss of epithelial phenotype and a gain in tumor cell proliferation and invasion. It has been hypothesized that cadherin-11 may be a molecular marker for a more aggressive subtype of breast cancer. The present study examined the expression of the mesenchymal gene/protein cadherin-11 in malignant, benign and healthy breast cancer samples. A paraffin-embedded tissue microarray of both malignant and benign/healthy breast tumor was used. Clinicopathological parameters, including age, grading, tumor size, hormone receptors and HER2 receptors status were obtained from patient medical records. Expression of cadherin-11 was analyzed using the monoclonal mouse anti cadherin-11 IgG2B clone. Total RNA was extracted from each breast cancer sample and subjected to semi-quantitative RT-PCR analysis for cadherin-11. Cadherin-11 was detected in 80/82 malignant breast cancer samples and in 33/70 non-malignant tissue samples. Cadherin-11 expression was observed to be predominantly localized to the membrane of tumor cells. When compared to healthy breast tissue biopsies, both cadherin-11 mRNA and protein were demonstrated to be significantly overexpressed in breast carcinoma (P=0.040 and P<0.0001, respectively). Within malignant tumors, however, protein expression was not identified to be associated with other clinicopathological parameters. Our results indicate that cadherin-11 expression is upregulated in malignant human breast cancer. PMID:28101202

  14. Gene Expression in Human Accessory Lacrimal Glands of Wolfring

    PubMed Central

    Ubels, John L.; Gipson, Ilene K.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Van Dyken, Rachel E.; Hatton, Mark P.

    2012-01-01

    Purpose. The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. Methods. Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. Results. Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. Conclusions. The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs. PMID:22956620

  15. Expression of the beta 7 integrin by human endothelial cells.

    PubMed Central

    Brezinschek, R. I.; Brezinschek, H. P.; Lazarovits, A. I.; Lipsky, P. E.; Oppenheimer-Marks, N.

    1996-01-01

    Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells. Images Figure 2 Figure 4 Figure 6 Figure 7 PMID:8909254

  16. Comparing anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH) as predictors of ovarian function.

    PubMed

    Barad, David H; Weghofer, Andrea; Gleicher, Norbert

    2009-04-01

    We compared predictive values of anti-Müllerian hormone (AMH) and baseline FSH with respect to IVF cycle outcomes based on oocyte numbers retrieved and number of clinical pregnancies established. In 76 IVF cycles investigated, AMH was clearly superior in predicting IVF outcomes in comparison with FSH.

  17. Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production.

    PubMed

    Serafim, M K B; Duarte, A B G; Silva, G M; Souza, C E A; Magalhães-Padilha, D M; Moura, A A A; Silva, L D M; Campello, C C; Figueiredo, J R

    2015-04-01

    Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Regulation of mda-7 gene expression during human melanoma differentiation.

    PubMed

    Madireddi, M T; Dent, P; Fisher, P B

    2000-03-02

    Induction of irreversible growth arrest and terminal differentiation in human melanoma cells following treatment with recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expression in differentiating human melanoma cells. The mda-7 gene is actively transcribed in uninduced HO-1 human melanoma cells and the rate of transcription of mda-7 is not significantly enhanced by treatment with IFN-beta, MEZ or IFN-beta+MEZ. The high basal activity of the mda-7 promoter in uninduced melanoma cells and the absence of enhancing effect upon treatment with differentiation inducers is corroborated by transfection studies using the promoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT - PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-beta+MEZ and to a lesser degree in MEZ treated cells. We show that induction of terminal differentiation of HO-1 cells with IFN-beta+MEZ dramatically increases the half-life of mda-7 mRNA while treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitutive activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda-7 expression during terminal differentiation. The 3' UTR region of mda-7 contains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled by a labile protein factor(s). Substitution of the SV40 polyadenylation signal sequence in the luciferase reporter plasmid with

  19. In situ expression of cytokines in human heart allografts.

    PubMed

    Van Hoffen, E; Van Wichen, D; Stuij, I; De Jonge, N; Klöpping, C; Lahpor, J; Van Den Tweel, J; Gmelig-Meyling, F; De Weger, R

    1996-12-01

    Although allograft rejection, the major complication of human organ transplantation, has been extensively studied, little is known about the exact cellular localization of the cytokine expression inside the graft during rejection. Therefore, we used in situ hybridization and immunohistochemistry to study local cytokine mRNA and protein expression in human heart allografts, in relation to the phenotypical characteristics of the cellular infiltrate. Clear expression of mRNA for interleukin (IL)-6, IL-8, IL-9, and IL-10 and weak expression for IL-2, IL-4, IL-5, and tumor necrosis factor (TNF)-alpha was detected in biopsies exhibiting high rejection grades (grade 3A/B). Also at lower grades of rejection, mRNA for IL-6 and IL-9 was present. Some mRNA for IL-1 beta, TNF-beta, and interferon (IFN)-gamma was detected in only a few biopsies. Using immunohistochemistry, IL-2, IL-3, and IL-10 protein was detected in biopsies with high rejection grades, whereas few cells expressed IL-6, IL-8, and IFN-gamma. In biopsies with lower grades of rejection, a weaker expression of these cytokines was observed. IL-4 was hardly detected in any of the biopsies. The level of IL-12 expression was equal in all biopsies. Although mRNA expression of several cytokines was expressed at a low level compared with the protein level of those cytokines, there was a good correlation between localization of cytokine mRNA and protein. Expression of IL-2, IL-4, IL-5, TNF-alpha, and IFN-gamma was mainly detected in lymphocytes. IL-3, IL-6, IL-10, and IL-12 were not detected or not only detected in lymphocytes but also in other stromal elements (eg, macrophages). Macrophage production of IL-3 and IL-12 was confirmed by immunofluorescent double labeling with CD68. We conclude that cardiac allograft rejection is not simply regulated by T helper cell cytokine production, but other intragraft elements contribute considerably to this process.

  20. Transgenic rabbit that expresses a functional human lipoprotein (a)

    DOEpatents

    Rouy, Didier; Duverger, Nicolas; Emmanuel, Florence; Denefle, Patrice; Houdebine, Louis-Marie; Viglietta, Celine; Rubin, Edward M.; Hughes, Steven D.

    2003-01-01

    A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

  1. Human myometrial gene expression before and during parturition.

    PubMed

    Havelock, Jon C; Keller, Patrick; Muleba, Ndaya; Mayhew, Bobbie A; Casey, Brian M; Rainey, William E; Word, R Ann

    2005-03-01

    Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition. The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes believed to be important in parturition. Fundal myometrial mRNA was isolated from five women in labor and five women not in labor, and analyzed using human UniGEM-V microarrays with 9182 cDNA elements. Real-time polymerase chain reaction using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes; namely, prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR). The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined. Immunolocalization of cyclooxygenase-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry. Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor. However, there appears to be a subset of genes important in the parturition cascade. The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of women in labor suggest that this protein may be very important in the initiation or propagation of human labor.

  2. Identification of Haemophilus ducreyi genes expressed during human infection.

    PubMed

    Bauer, Margaret E; Fortney, Kate R; Harrison, Alistair; Janowicz, Diane M; Munson, Robert S; Spinola, Stanley M

    2008-04-01

    To identify Haemophilus ducreyi transcripts that are expressed during human infection, we used selective capture of transcribed sequences (SCOTS) with RNA isolated from pustules obtained from three volunteers infected with H. ducreyi, and with RNA isolated from broth-grown bacteria used to infect volunteers. With SCOTS, competitive hybridization of tissue-derived and broth-derived sequences identifies genes that may be preferentially expressed in vivo. Among the three tissue specimens, we identified 531 genes expressed in vivo. Southern blot analysis of 60 genes from each tissue showed that 87 % of the identified genes hybridized better with cDNA derived from tissue specimens than with cDNA derived from broth-grown bacteria. RT-PCR on nine additional pustules confirmed in vivo expression of 10 of 11 selected genes in other volunteers. Of the 531 genes, 139 were identified in at least two volunteers. These 139 genes fell into several functional categories, including biosynthesis and metabolism, regulation, and cellular processes, such as transcription, translation, cell division, DNA replication and repair, and transport. Detection of genes involved in anaerobic and aerobic respiration indicated that H. ducreyi likely encounters both microenvironments within the pustule. Other genes detected suggest an increase in DNA damage and stress in vivo. Genes involved in virulence in other bacterial pathogens and 32 genes encoding hypothetical proteins were identified, and may represent novel virulence factors. We identified three genes, lspA1, lspA2 and tadA, known to be required for virulence in humans. This is the first study to broadly define transcripts expressed by H. ducreyi in humans.

  3. Transcriptional regulation of human thromboxane synthase gene expression

    SciTech Connect

    Lee, K.D.; Baek, S.J.; Fleischer, T

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  4. Changes in Gene Expression in Human Meibomian Gland Dysfunction

    PubMed Central

    Liu, Shaohui; Richards, Stephen M.; Lo, Kristine; Hatton, Mark; Fay, Aaron

    2011-01-01

    Purpose. Meibomian gland dysfunction (MGD) may be the leading cause of dry eye syndrome throughout the world. However, the precise mechanism(s) underlying the pathogenesis of this disease is unclear. This study was conducted to identify meibomian gland genes that may promote the development and/or progression of human MGD. Methods. Lid tissues were obtained from male and female MGD patients and age-matched controls after eyelid surgeries (e.g., to correct entropion or ectropion). Meibomian glands were isolated and processed for RNA extraction and the analysis of gene expression. Results. The results show that MGD is associated with significant alterations in the expression of almost 400 genes in the human meibomian gland. The levels of 197 transcripts, including those encoding various small proline-rich proteins and S100 calcium-binding proteins, are significantly increased, whereas the expression of 194 genes, such as claudin 3 and cell adhesion molecule 1, is significantly decreased. These changes, which cannot be accounted for by sex differences, are accompanied by alterations in many gene ontologies (e.g., keratinization, cell cycle, and DNA repair). The findings also show that the human meibomian gland contains several highly expressed genes that are distinct from those in an adjacent tissue (i.e., conjunctival epithelium). Conclusions. The results demonstrate that MGD is accompanied by multiple changes in gene expression in the meibomian gland. The nature of these alterations, including the upregulation of genes encoding small proline-rich proteins and S100 calcium-binding proteins, suggest that keratinization plays an important role in the pathogenesis of MGD. PMID:21372006

  5. Human pyridoxal phosphatase. Molecular cloning, functional expression, and tissue distribution.

    PubMed

    Jang, Young Min; Kim, Dae Won; Kang, Tae-Cheon; Won, Moo Ho; Baek, Nam-In; Moon, Byung Jo; Choi, Soo Young; Kwon, Oh-Shin

    2003-12-12

    Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 microM and 1.52 s(-1), respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

  6. Genetic variations altering FSH action affect circulating hormone levels as well as follicle growth in healthy peripubertal girls.

    PubMed

    Busch, Alexander S; Hagen, Casper P; Almstrup, Kristian; Main, Katharina M; Juul, Anders

    2016-04-01

    counts, however, a cumulative minor allele count (FSHB c.-211 G>T and FSHR c.-29G>A) was negatively associated with the number of large follicles (≥5 mm) (n = 91, P = 0.04) (adjusted for Tanner stages). Since we studied girls and young adolescents during pubertal transition, our study may not be fully comparable with previous studies on FSHB and FSHR variants in adult women. The group of young adolescents (Tanner B4 + B5) reflects the endocrine situation in adult women best, however, the group is not large enough to contribute substantially to the conflicting results concerning the influence of FSHB c.-211G>T in adult women. Furthermore, we have no information about the exact day of the menstrual cycle in the subgroup of girls with menarche. The sex-specific interaction of FSHB and FSHR genetic variants and physiological as well as pathological conditions is being increasingly elucidated. The variant triplet set might serve as diagnostic and pharmacogenetic marker. For the first time, we show an additional effect of FSHR c.-29G>A on serum FSH levels in healthy girls. Moreover, morphological data suggest impaired FSH-induced maturation of ovarian follicles in minor allele carriers of FSHB c.-211G>T and FSHR c.-29G>A. This may explain previous findings of delayed pubertal onset in these girls. Funding was provided by the Danish Agency for Science, Technology and Innovation (09-067180), Danish Ministry of the Environment, CeHoS (MST-621-00065), Capital Region of Denmark (December 2011), Ministry of Higher Education and Science (DFF-1331-00113) and EDMaRC (Danish Ministry of Health). A.S.B. was funded from December 2015 by ReproUnion (EU Interreg Öresund-Kattegat-Skagerrak). The authors declare no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

    PubMed Central

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-01-01

    ABSTRACT Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR. PMID:28791335

  8. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

    PubMed

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-06-01

    Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn(56) of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

  9. Study of human dopamine sulfotransferases based on gene expression programming.

    PubMed

    Si, Hongzong; Zhao, Jiangang; Cui, Lianhua; Lian, Ning; Feng, Hanlin; Duan, Yun-Bo; Hu, Zhide

    2011-09-01

    A quantitative model is developed to predict the Km of 47 human dopamine sulfotransferases by gene expression programming. Each kind of compound is represented by several calculated structural descriptors of moment of inertia A, average electrophilic reactivity index for a C atom, relative number of triple bonds, RNCG relative negative charge, HA-dependent HDSA-1, and HBCA H-bonding charged surface area. Eight fitness functions of the gene expression programming method are used to find the best nonlinear model. The best quantitative model with squared standard error and square of correlation coefficient are 0.096 and 0.91 for training data set, and 0.102 and 0.88 for test set, respectively. It is shown that the gene expression programming-predicted results with fitness function are in good agreement with experimental ones. © 2011 John Wiley & Sons A/S.

  10. Doublecortin expression in the normal and epileptic adult human brain.

    PubMed

    Liu, Y W J; Curtis, M A; Gibbons, H M; Mee, E W; Bergin, P S; Teoh, H H; Connor, B; Dragunow, M; Faull, R L M

    2008-12-01

    Mesial temporal lobe epilepsy (MTLE) is a neurological disorder associated with spontaneous recurrent complex partial seizures and hippocampal sclerosis. Although increased hippocampal neurogenesis has been reported in animal models of MTLE, increased neurogenesis has not been reported in the hippocampus of adult human MTLE cases. Here we showed that cells expressing doublecortin (Dcx), a microtubule-associated protein expressed in migrating neuroblasts, were present in the hippocampus and temporal cortex of the normal and MTLE adult human brain. In particular, increased numbers of Dcx-positive cells were observed in the epileptic compared with the normal temporal cortex. Importantly, 56% of Dcx-expressing cells in the epileptic temporal cortex coexpressed both the proliferative cell marker, proliferating cell nuclear antigen and early neuronal marker, TuJ1, suggesting that they may be newly generated neurons. A subpopulation of Dcx-positive cells in the epileptic temporal cortex also coexpressed the mature neuronal marker, NeuN, suggesting that epilepsy may promote the generation of new neurons in the temporal cortex. This study has identified, for the first time, a novel population of Dcx-positive cells in the adult human temporal cortex that can be upregulated by epilepsy and thus, raises the possibility that these cells may have functional significance in the pathophysiology of epilepsy.

  11. Biologic and Therapeutic Significance of MYB Expression in Human Melanoma

    NASA Astrophysics Data System (ADS)

    Hijiya, Nobuko; Zhang, Jin; Ratajczak, Mariusz Z.; Kant, Jeffrey A.; Deriel, Kim; Herlyn, Meenhard; Zon, Gerald; Gewirtz, Alan M.

    1994-05-01

    We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides. Inhibition of cell growth correlated with specific decrease of MYB mRNA. In SCID mice bearing human melanoma tumors, infusion of MYB antisense transiently suppressed MYB gene expression but effected long-term growth suppression of transplanted tumor cells. Toxicity of the oligodeoxynucleotides was minimal in mice, even when targeted to the murine Myb gene. These results suggest that the MYB gene may play an important, though undefined, role in the growth of at least some human melanomas. Inhibition of MYB expression might be of use in the treatment of this disease.

  12. Restoration of Vision with Ectopic Expression of Human Rod Opsin.

    PubMed

    Cehajic-Kapetanovic, Jasmina; Eleftheriou, Cyril; Allen, Annette E; Milosavljevic, Nina; Pienaar, Abigail; Bedford, Robert; Davis, Katherine E; Bishop, Paul N; Lucas, Robert J

    2015-08-17

    Many retinal dystrophies result in photoreceptor loss, but the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. Here, we show that ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, can function outside native photoreceptors and restore visual function in a mouse model of advanced retinal degeneration. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing (increases and decreases) induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioral responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance (≈50-100 lux) typical of natural indoor environments. These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Expression of pemphigus-autoantigen desmoglein 1 in human thymus.

    PubMed

    Mouquet, H; Berrih-Aknin, S; Bismuth, J; Joly, P; Gilbert, D; Tron, F

    2008-05-01

    Desmoglein (Dsg) 1 is a transmembrane glycoprotein of the desmosome allowing cell-cell adhesion between keratinocytes, whose expression is restricted to stratified squamous epithelia-like epidermis. Dsg1 is the target autoantigen of pathogenic autoantibodies produced by pemphigus foliaceus and 50% of pemphigus vulgaris patients in a Dsg1-specific T-cell-dependent pathway. Herewith, we show that mRNA of the DSG1 gene is present in normal human thymus and show by quantitative real-time polymerase chain reaction analysis that the expression of DSG1 transcript increases with age. Although immunoblot analysis on human thymus extracts using different anti-Dsg1 antibodies did not allow to detect the protein, we show by double-immunofluorescence assay that Dsg1 is expressed at protein level by CD19+ CD63+ cells located in the medulla. These data provide another illustration of the thymic expression of a tissue-specific autoantigen involved in an organ-specific autoimmune disease, which may participate in the tolerance acquisition and/or regulation of Dsg1-specific T cells.

  14. Expression of 5-Lipoxygenase in human colorectal cancer

    PubMed Central

    Soumaoro, Labile Togba; Iida, Satoru; Uetake, Hiroyuki; Ishiguro, Megumi; Takagi, Yoko; Higuchi, Tetsuro; Yasuno, Masamichi; Enomoto, Masayuki; Sugihara, Kenichi

    2006-01-01

    AIM: To evaluate the 5-lipoxygenases (Loxs) expression level in human colorectal cancer specimens in order to determine its clinicopathologic significance in human tumorigenesis. METHODS: The relative quantity of 5-Lox mRNA in paired 91 colorectal tumor and adjacent normal mucosa samples was determined by real time quantitative PCR. Additionally, the expression of 5-Lox and cyclooxygenase (Cox)-2 proteins was also examined using immunohistochemical staining methods. RESULTS: There was a marked increase in 5-Lox mRNA levels in the tumor compared with paired normal mucosa samples (P < 0.0001). Sixty six (72.5%) tumors showed high 5-Lox mRNA levels. The positivity rate of 5-Lox and Cox-2 protein expression was 68.7% and 79.1% respectively. There was a significant association between tumoral 5-Lox mRNA level and tumor size (Rho = 0.392, P = 0.0002), depth or vessel invasion. CONCLUSION: These results suggest that 5-Lox is up-regulated in colorectal cancer and that inhibition of its expression might be valuable in the prevention and treatment of colorectal cancer. PMID:17072961

  15. miRNA Expression in Pediatric Failing Human Heart

    PubMed Central

    Stauffer, Brian L.; Russell, Gloria; Nunley, Karin; Miyamoto, Shelley D.; Sucharov, Carmen C.

    2013-01-01

    miRNAs are short regulatory RNAs that can regulate gene expression through interacting with the 3'UTR of target mRNAs. Although the role of miRNAs has been extensively studied in adult human and animal models of heart disease, nothing is known about their expression in pediatric heart failure patients. Different than adults with heart failure, pediatric patients respond well to phosphodiesterase inhibitor (PDEi) treatment, which is safe in the outpatient setting, results in fewer heart failure emergency department visits, fewer cardiac hospital admissions and improved NYHA classification. We have recently shown that the pediatric heart failure patients display a unique molecular profile that is different from adults with heart failure. In this study we show for the first time that pediatric heart failure patients display a unique miRNA profile, and that expression of some miRNAs correlate with response to PDEi treatment. Moreover, we show that expression of Smad4, a potential target for PDEi-regulated miRNAs, is normalized in PDEi-treated patients. Since miRNAs may be used as therapy for human heart failure, our results underscore the importance of defining the molecular characteristics of pediatric heart failure patients, so age-appropriate therapy can be designed for this population. PMID:23333438

  16. Comparative expression pathway analysis of human and canine mammary tumors

    PubMed Central

    Uva, Paolo; Aurisicchio, Luigi; Watters, James; Loboda, Andrey; Kulkarni, Amit; Castle, John; Palombo, Fabio; Viti, Valentina; Mesiti, Giuseppe; Zappulli, Valentina; Marconato, Laura; Abramo, Francesca; Ciliberto, Gennaro; Lahm, Armin; La Monica, Nicola; de Rinaldis, Emanuele

    2009-01-01

    Background Spontaneous tumors in dog have been demonstrated to share many features with their human counterparts, including relevant molecular targets, histological appearance, genetics, biological behavior and response to conventional treatments. Mammary tumors in dog therefore provide an attractive alternative to more classical mouse models, such as transgenics or xenografts, where the tumour is artificially induced. To assess the extent to which dog tumors represent clinically significant human phenotypes, we performed the first genome-wide comparative analysis of transcriptional changes occurring in mammary tumors of the two species, with particular focus on the molecular pathways involved. Results We analyzed human and dog gene expression data derived from both tumor and normal mammary samples. By analyzing the expression levels of about ten thousand dog/human orthologous genes we observed a significant overlap of genes deregulated in the mammary tumor samples, as compared to their normal counterparts. Pathway analysis of gene expression data revealed a great degree of similarity in the perturbation of many cancer-related pathways, including the 'PI3K/AKT', 'KRAS', 'PTEN', 'WNT-beta catenin' and 'MAPK cascade'. Moreover, we show that the transcriptional relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Conclusion Our data confirm and further strengthen the value of the canine mammary cancer model and open up new perspectives for the evaluation of novel cancer therapeutics and the development of prognostic and diagnostic biomarkers to be used in clinical studies. PMID:19327144

  17. Han Chinese polycystic ovary syndrome risk variants in women of European ancestry: relationship to FSH levels and glucose tolerance.

    PubMed

    Saxena, R; Georgopoulos, N A; Braaten, T J; Bjonnes, A C; Koika, V; Panidis, D; Welt, C K

    2015-06-01

    associated with insulin (-0.16 ± 0.05, P = 0.0029) and glucose levels (-0.20 ± 0.05, P = 0.0002) 120 min after an oral glucose test. The study was large and contained replication cohorts, but was limited by a small number of controls in the Greek cohort and a small number of cases in the second Boston cohort. The second Boston group was identified using electronic medical record review, but was validated for the cardinal features of PCOS. This study demonstrates a cross-ethnic PCOS risk locus in FSHR in women of European ancestry with PCOS. The variant may influence FSH receptor responsiveness as suggested by the associated change in FSH levels. The relationship between a variant near RAB5B and SUOX and glucose stimulated insulin and glucose levels suggests an influence of one of these genes on glucose tolerance, but the absence of a relationship with PCOS points to potential differences in the international PCOS patient populations. The project was supported by Award Number R01HD065029 from the Eunice Kennedy Shriver National Institute Of Child Health & Human Development, Award Number 1 UL1 RR025758, Harvard Clinical and Translational Science Center, from the National Center for Research Resources, award 1-10-CT-57 from the American Diabetes Association and the Partners Healthcare Center for Personalized Genetics Project Grant. C.K.W. is a consultant for Takeda Pharmaceuticals. NCT00166569. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Connexin expression in nonneoplastic human prostate epithelial cells.

    PubMed

    Saladino, Francesca; Carruba, Giuseppe; Quader, Salmaan T A; Amoroso, Maria; Di Cristina, Antoniette; Webber, Mukta M; Castagnetta, Luigi A M

    2002-06-01

    Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.

  19. Expression and clinical value of EGFR in human meningiomas

    PubMed Central

    Backer-Grøndahl, Thomas; Ytterhus, Borgny; Granli, Unn S.; Lydersen, Stian; Gulati, Sasha; Torp, Sverre H.

    2017-01-01

    Background Meningiomas are common intracranial tumors in humans that frequently recur despite having a predominantly benign nature. Even though these tumors have been shown to commonly express EGFR/c-erbB1 (epidermal growth factor receptor), results from previous studies are uncertain regarding the expression of either intracellular or extracellular domains, cellular localization, activation state, relations to malignancy grade, and prognosis. Aims This study was designed to investigate the expression of the intracellular and extracellular domains of EGFR and of the activated receptor as well as its ligands EGF and TGFα in a large series of meningiomas with long follow-up data, and investigate if there exists an association between antibody expression and clinical and histological data. Methods A series of 186 meningiomas consecutively operated within a 10-year period was included. Tissue microarrays were constructed and immunohistochemically analyzed with antibodies targeting intracellular and extracellular domains of EGFR, phosphorylated receptor, and EGF and TGFα. Expression levels were recorded as a staining index (SI). Results Positive immunoreactivity was observed for all antibodies in most cases. There was in general high SIs for the intracellular domain of EGFR, phosphorylated EGFR, EGF, and TGFα but lower for the extracellular domain. Normal meninges were negative for all antibodies. Higher SIs for the phosphorylated EGFR were observed in grade II tumors compared with grade I (p = 0.018). Survival or recurrence was significantly decreased in the time to recurrence analysis (TTR) with high SI-scores of the extracellular domain in a univariable survival analysis (HR 1.152, CI (1.036–1.280, p = 0.009)). This was not significant in a multivariable analysis. Expression of the other antigens did not affect survival. Conclusion EGFR is overexpressed and in an activated state in human meningiomas. High levels of ligands also support this growth factor

  20. Development of an homologous radioimmunoassay for chicken follicle-stimulating hormone and measurement of plasma FSH during the ovulatory cycle.

    PubMed

    Krishnan, K A; Proudman, J A; Bolt, D J; Bahr, J M

    1993-08-01

    1. A highly specific and sensitive homologous radioimmunoassay was developed for measurement of chicken follicle stimulating hormone (cFSH). 2. Mammalian gonadotropin releasing hormone (GnRH) significantly stimulated secretion of chicken luteinising hormone (cLH) but not cFSH when administered to 22 week non-laying hens. 3. Chicken GnRH-I did not affect circulating cFSH concentrations but significantly stimulated cLH secretion when administered to 3 week cockerels. 4. The plasma concentration of cFSH was low throughout the ovulatory cycle, but a significant decline in cFSH occurred prior to the pre-ovulatory LH surge and a significant increase occurred during the 3 hr prior to oviposition as LH levels decline.

  1. Novel FSHβ mutation in a male patient with isolated FSH deficiency and infertility.

    PubMed

    Zheng, Junjie; Mao, Jiangfeng; Cui, Mingxuan; Liu, Zhaoxiang; Wang, Xi; Xiong, Shuyu; Nie, Min; Wu, Xueyan

    2017-06-01

    Isolated follicle stimulating hormone (FSH) deficiency due to mutations in FSHβ is an extremely rare autosomal recessive disease that has only been reported in ten patients to date. Symptoms of the disease include amenorrhoea and hypogonadism in women and azoospermia and normal testosterone levels in men. This study describes a Chinese male patient who presented with cryptorchidism and infertility. His serum hormonal profile revealed low FSH, elevated LH and normal testosterone levels. Sequence analysis identified a novel homozygous mutation in the FSHβ gene (c.343C > T) predicted to result in a premature termination codon and a truncated FSH protein (p.R115X). Both parents were heterozygous carriers of the mutation with normal pubertal development and fertility. The patient's testicular volume increased after one year of exogenous FSH replacement therapy at which point spermatocytes were detected in seminal samples, indicating potential future spermatogenesis. The expanded spectrum of FSHβ mutations and associated clinical manifestations described in this study may improve the diagnosis and treatment of this disease. Copyright © 2017. Published by Elsevier Masson SAS.

  2. Weight gain increases human aromatase expression in mammary gland.

    PubMed

    Chen, Dong; Zhao, Hong; Coon, John S; Ono, Masanori; Pearson, Elizabeth K; Bulun, Serdar E

    2012-05-15

    Adulthood weight gain predicts estrogen receptor-positive breast cancer. Because local estrogen excess in the breast likely contributes to cancer development, and aromatase is the key enzyme in estrogen biosynthesis, we investigated the role of local aromatase expression in weight gain-associated breast cancer risk in a humanized aromatase (Arom(hum)) mouse model containing the coding region and the 5'-regulatory region of the human aromatase gene. Compared with littermates on normal chow, female Arom(hum) mice on a high fat diet gained more weight, and had a larger mammary gland mass with elevated total human aromatase mRNA levels via promoters I.4 and II associated with increased levels of their regulators TNFα and C/EBPβ. There was no difference in total human aromatase mRNA levels in gonadal white adipose tissue. Our data suggest that diet-induced weight gain preferentially stimulates local aromatase expression in the breast, which may lead to local estrogen excess and breast cancer risk.

  3. Human platelets express authentic CB₁ and CB₂ receptors.

    PubMed

    Catani, M V; Gasperi, V; Catanzaro, G; Baldassarri, S; Bertoni, A; Sinigaglia, F; Avigliano, L; Maccarrone, M

    2010-11-01

    In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB₁) or type-2 (CB₂) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB₁ and CB₂ are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB₁, and to a lesser extent CB₂, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB₁ and CB₂ in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

  4. Evidence that lamprey GnRH-III does not release FSH selectively in cattle.

    PubMed

    Amstalden, M; Zieba, D A; Garcia, M R; Stanko, R L; Welsh, T H; Hansel, W H; Williams, G L

    2004-01-01

    Experiments were performed to test the hypothesis that lamprey GnRH-III (lGnRH-III) selectively releases FSH. Primary cultures of bovine adenohypophyseal cells were treated with mammalian GnRH (mGnRH) and lGnRH-III (10(-9), 10(-8), 10(-7) and 10(-6) M) or control media in Experiment 1. All doses of mGnRH and the two highest doses of lGnRH-III stimulated (P < 0.001) a non-selective release of LH and FSH. In Experiments 2-4, Latin Square designs were utilized in vivo to examine whether physiological and hormonal milieu regulate putative selective effects of lGnRH-III. In Experiments 2 and 3, ovariectomized cows with basal levels of estradiol only (Experiment 2) or in combination with luteal phase levels of progester-one (Experiment 3) were injected with mGnRH and lGnRH-III (0.055, 0.11, 0.165 and 1.1 microg/kg body weight (BW) and saline. All doses of mGnRH released (P < 0.001) LH and FSH, but only the highest dose of lGnRH-III stimulated (P < 0.001) a non-selective release of both LH and FSH (Experiment 3). For Experiments 4A and 4B, intact, mid-luteal phase cows were injected with mGnRH and lGnRH-III (1.1 microg/kg BW; Experiment 4A), lGnRH-III (1.1 and 4.4 microg/kg BW; Experiment 4B) and saline. As before, mGnRH released (P < 0.001) both LH and FSH at all doses. In contrast, lGnRH-III at the highest dose released (P < 0.001) LH but not FSH. These findings suggest that lGnRH-III may act as a weak competitor for the mGnRH receptor and do not support the hypothesis that it selectively releases FSH in cattle.

  5. Polycythemia in transgenic mice expressing the human erythropoietin gene

    SciTech Connect

    Semenza, G.L.; Traystman, M.D.; Gearhart, J.D.; Antonarakis, S.E. )

    1989-04-01

    Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5{prime} flanking sequence and 0.7 kilobase of 3{prime} flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.

  6. Cloning, functional expression and characterization of a human olfactory receptor.

    PubMed

    Hatt, H; Gisselmann, G; Wetzel, C H

    1999-05-01

    The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.

  7. Human enteric defensins. Gene structure and developmental expression.

    PubMed

    Mallow, E B; Harris, A; Salzman, N; Russell, J P; DeBerardinis, R J; Ruchelli, E; Bevins, C L

    1996-02-23

    Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5'-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19 24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern

  8. High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

    PubMed Central

    Chng, Zhenzhi; Peh, Gary S. L.; Herath, Wishva B.; Cheng, Terence Y. D.; Ang, Heng-Pei; Toh, Kah-Peng; Robson, Paul; Mehta, Jodhbir S.; Colman, Alan

    2013-01-01

    Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. PMID:23844023

  9. Differentially Expressed Genes and Signature Pathways of Human Prostate Cancer

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Robbins, Charles J.; Sang, Qing-Xiang Amy

    2015-01-01

    Genomic technologies including microarrays and next-generation sequencing have enabled the generation of molecular signatures of prostate cancer. Lists of differentially expressed genes between malignant and non-malignant states are thought to be fertile sources of putative prostate cancer biomarkers. However such lists of differentially expressed genes can be highly variable for multiple reasons. As such, looking at differential expression in the context of gene sets and pathways has been more robust. Using next-generation genome sequencing data from The Cancer Genome Atlas, differential gene expression between age- and stage- matched human prostate tumors and non-malignant samples was assessed and used to craft a pathway signature of prostate cancer. Up- and down-regulated genes were assigned to pathways composed of curated groups of related genes from multiple databases. The significance of these pathways was then evaluated according to the number of differentially expressed genes found in the pathway and their position within the pathway using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. The “transforming growth factor-beta signaling” and “Ran regulation of mitotic spindle formation” pathways were strongly associated with prostate cancer. Several other significant pathways confirm reported findings from microarray data that suggest actin cytoskeleton regulation, cell cycle, mitogen-activated protein kinase signaling, and calcium signaling are also altered in prostate cancer. Thus we have demonstrated feasibility of pathway analysis and identified an underexplored area (Ran) for investigation in prostate cancer pathogenesis. PMID:26683658

  10. FSH withdrawal improves developmental competence of oocytes in the bovine model.

    PubMed

    Nivet, Anne-Laure; Bunel, Audrey; Labrecque, Rémi; Belanger, Josée; Vigneault, Christian; Blondin, Patrick; Sirard, Marc-André

    2012-02-01

    Combinations of genetic, environmental, and management factors are suspected to explain the loss in fertility observed for over 20 years in dairy cows. In some cases, IVF is used. When compared with in vivo embryo production, IVF resulted in low success rates until the FSH coasting process (FSH starvation after superstimulation) was introduced in 2002. Increased competence associated with FSH withdrawal of aspirated oocyte for in vitro maturation and IVF has not been optimized nor explained yet. The goal here was to determine and characterize the optimal oocyte competence acquisition window during the coasting period by determining blastocyst rates and follicular cohort development. Commercial milking cycling cows (n=6) were stimulated with 3 days of FSH (6×40 mg NIH Folltropin-V given at 12 h intervals) followed by a coasting period of 20, 44, 68, or 92 h. Each animal was exposed to the four conditions and served as its own control. At the scheduled time, transvaginal aspirations of immature oocytes were performed followed by IVF of half the oocytes. The outcomes were as follows: i) FSH coasting was optimal at a defined period: between 44 and 68 h of coasting; ii) The best estimated coasting duration was ∼54±7 h; iii) Under these conditions, the best statistical blastocyst rate estimation was ∼70%; iv) Between 44 and 68 h of coasting, follicle size group proportions were similar; v) Follicle diameter was not linearly associated with competence. In conclusion, coasting duration is critical to harvest the oocytes at the right moment of follicular differentiation.

  11. Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract

    PubMed Central

    Porter, Edith; Bevins, Charles L.; DiRosario, Julianne; Becknell, Brian; Wang, Huanyu

    2012-01-01

    Background The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects. Methodology/Principal Findings Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection. Conclusions/Significance DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility. PMID:22359618

  12. Human articular chondrocytes express functional leukotriene B4 receptors

    PubMed Central

    Hansen, Ann Kristin; Indrevik, Jill-Tove; Figenschau, Yngve; Martinez-Zubiaurre, Inigo; Sveinbjörnsson, Baldur

    2015-01-01

    Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified. PMID:25677035

  13. Increased FNDC5/Irisin expression in human hepatocellular carcinoma.

    PubMed

    Gaggini, Melania; Cabiati, Manuela; Del Turco, Serena; Navarra, Teresa; De Simone, Paolo; Filipponi, Franco; Del Ry, Silvia; Gastaldelli, Amalia; Basta, Giuseppina

    2017-02-01

    The fibronectin type III domain containing 5 (FNDC5)/Irisin, a novel energy-regulating hormone, is associated with lipid and carbohydrate metabolism. It is produced in low amounts by normal hepatic tissue, while in human hepatocellular carcinoma (HCC), in which aberrant de novo lipogenesis (DNL) occurs, the hepatic expression of FNDC5/Irisin is still unknown. The gene expression of FNDC5/Irisin, associated to key regulators of DNL, inflammation and cancer progression was evaluated in liver tissue of 18 patients with HCC undergoing liver transplantation and of 18 deceased donors. Hepatic mRNA expression of FNDC5/Irisin and stearoyl-CoA desaturase (SCD-1), main enzymatic regulator of DNL, were significantly higher in HCC patients than in donors (p<0.0001 and p=0.015, respectively). The hepatic mRNA expression of the neurogenic locus notch homolog protein 1 (NOTCH1) tended to be higher in HCC patients than in donors (p=0.06). Only in HCC patients, hepatic FNDC5/Irisin strongly correlated with the transcription factor sterol regulatory element-binding factor 1, SCD-1, NOTCH1, tumor necrosis factor-α and Interleukin-6 mRNA expression. Further, in HCC patients, FNDC5/Irisin mRNA tended to correlate to plasma lipid profile namely triglycerides, palmitic/linoleic acid and polyunsaturated fatty acid/saturated fatty acid ratios. In conclusion, HCC-liver tissue over-expressed FNDC5/Irisin in association with gene expression of mediators involved in lipogenesis, inflammation and cancer, suggesting a possible protective role of the hormone from the liver damage.

  14. Determinants of human plasma glutathione peroxidase (GPx-3) expression.

    PubMed

    Bierl, Charlene; Voetsch, Barbara; Jin, Richard C; Handy, Diane E; Loscalzo, Joseph

    2004-06-25

    Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 deficiency has been associated with cardiovascular disease and stroke. The regulation of GPx-3 expression remains largely uncharacterized, however, and we studied its transcriptional and translational determinants in a cultured cell system. In transient transfections of a renal cell line (Caki-2), the published sequence cloned upstream of a luciferase reporter gene produced minimal activity (relative luminescence (RL) = 0.6 +/- 0.4). Rapid amplification of cDNA ends was used to identify a novel transcription start site that is located 233 bp downstream (3') of the published site and that produced a >25-fold increase in transcriptional activity (RL = 16.8 +/- 1.9; p < 0.0001). Analysis of the novel GPx-3 promoter identified Sp-1- and hypoxia-inducible factor-1-binding sites, as well as the redox-sensitive metal response element and antioxidant response element. Hypoxia was identified as a strong transcriptional regulator of GPx-3 expression, in part through the presence of the hypoxia-inducible factor-1-binding site, leading to an almost 3-fold increase in expression levels after 24 h compared with normoxic conditions (normalized RL = 3.5 +/- 0.3 versus 1.2 +/- 0.1; p < 0.001). We also investigated the role of the translational cofactors tRNA(Sec), SECIS-binding protein-2, and SelD (selenophosphate synthetase D) in GPx-3 protein expression. tRNA(Sec) and SelD significantly enhanced GPx-3 expression, whereas SECIS-binding protein-2 showed a trend toward increased expression. These results demonstrate the presence of a novel functional transcription start site for the human GPx-3 gene with a promoter regulated by hypoxia, and identify unique translational determinants of GPx-3 expression.

  15. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  16. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    NASA Technical Reports Server (NTRS)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  17. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    NASA Technical Reports Server (NTRS)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  18. Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

    PubMed

    Chen, Zhiheng; Tang, Yamei; Yang, Zuocheng; Liu, Shaojun; Liu, Yong; Li, Yan; He, Wei

    2013-09-01

    Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

  19. Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications

    PubMed Central

    PONIEWIERSKA-BARAN, AGATA; SUSZYNSKA, MALWINA; SUN, WENYUE; ABDELBASET-ISMAIL, AHMED; SCHNEIDER, GABRIELA; BARR, FREDERIC G.; RATAJCZAK, MARIUSZ Z.

    2015-01-01

    The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression. PMID:26412593

  20. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  1. CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

    PubMed Central

    Messadi, D. V.; Bertolami, C. N.

    1993-01-01

    Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-labeling immunohistochemistry using the two anti-CD44 monoclonal antibodies, BU-52 and J173, and a biotinylated hyaluronan binding complex probe, b-HABR. Certain cultures were pretreated with Streptomyces hyaluronidase to assess the dependency of CD44 expression on the presence of endogenous hyaluronan. CD44 expression, both in the presence and the absence of exogenous hyaluronan, was quantitated by radioimmunobinding assay. Overall glycosaminoglycan synthesis and identification of hyaluronan were accomplished by precursor incorporation assays and by quantitative cellulose acetate electrophoresis. CD44 was found to be a normal human adult fibroblastic antigen whose expression is markedly increased for hypertrophic scar fibroblasts compared with normal skin fibroblasts. Although hyaluronan was found to be the predominant glycosaminoglycan constituent of the pericellular matrix for these fibroblasts, CD44 attachment to the cell surface is neither mediated by hyaluronan nor is the presence of hyaluronan a prerequisite for CD44 expression. Exogenous hyaluronan induced a decline in measurable CD44 expression for normal skin fibroblasts but not for hypertrophic scar fibroblasts. These observations are compatible with current understanding of the way cells manage the hyaluronan economy of the extracellular matrix and emphasize phenotypic heterogeneities between fibroblasts derived from normal versus scar tissues. Images Figure 1 Figure 4 PMID:8475990

  2. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  3. Differentially expressed genes and canonical pathway expression in human atherosclerotic plaques – Tampere Vascular Study

    PubMed Central

    Sulkava, Miska; Raitoharju, Emma; Levula, Mari; Seppälä, Ilkka; Lyytikäinen, Leo-Pekka; Mennander, Ari; Järvinen, Otso; Zeitlin, Rainer; Salenius, Juha-Pekka; Illig, Thomas; Klopp, Norman; Mononen, Nina; Laaksonen, Reijo; Kähönen, Mika; Oksala, Niku; Lehtimäki, Terho

    2017-01-01

    Cardiovascular diseases due to atherosclerosis are the leading cause of death globally. We aimed to investigate the potentially altered gene and pathway expression in advanced peripheral atherosclerotic plaques in comparison to healthy control arteries. Gene expression analysis was performed (Illumina HumanHT-12 version 3 Expression BeadChip) for 68 advanced atherosclerotic plaques (15 aortic, 29 carotid and 24 femoral plaques) and 28 controls (left internal thoracic artery (LITA)) from Tampere Vascular Study. Dysregulation of individual genes was compared to healthy controls and between plaques from different arterial beds and Ingenuity pathway analysis was conducted on genes with a fold change (FC) > ±1.5 and false discovery rate (FDR) < 0.05. 787 genes were significantly differentially expressed in atherosclerotic plaques. The most up-regulated genes were osteopontin and multiple MMPs, and the most down-regulated were cell death-inducing DFFA-like effector C and A (CIDEC, CIDEA) and apolipoprotein D (FC > 20). 156 pathways were differentially expressed in atherosclerotic plaques, mostly inflammation-related, especially related with leukocyte trafficking and signaling. In artery specific plaque analysis 50.4% of canonical pathways and 41.2% GO terms differentially expressed were in common for all three arterial beds. Our results confirm the inflammatory nature of advanced atherosclerosis and show novel pathway differences between different arterial beds. PMID:28128285

  4. Transcription factor p53 can regulate proliferation, apoptosis and secretory activity of luteinizing porcine ovarian granulosa cell cultured with and without ghrelin and FSH.

    PubMed

    Sirotkin, A V; Benco, A; Tandlmajerova, A; Vasícek, D; Kotwica, J; Darlak, K; Valenzuela, F

    2008-11-01

    The aim of our in vitro experiments was to examine the role of transcription factor p53 in controlling the basic functions of ovarian cells and their response to hormonal treatments. Porcine ovarian granulosa cells, transfected and non-transfected with a gene construct encoding p53, were cultured with ghrelin and FSH (all at concentrations of 0, 1, 10, or 100 ng/ml). Accumulation of p53, of apoptosis-related (MAP3K5) and proliferation-related (cyclin B1) substances was evaluated by immunocytochemistry. The secretion of progesterone (P(4)), oxytocin (OT), prostaglandin F (PGF), and E (PGE) was measured by RIA. Transfection with the p53 gene construct promoted accumulation of this transcription factor within cells. It also stimulated the expression of a marker of apoptosis (MAP3K5). Over-expression of p53 resulted in reduced accumulation of a marker of proliferation (cyclin B1), P(4), and PGF secretion and increased OT and PGE secretion. Ghrelin, when added alone, did not affect p53 or P(4), but reduced MAP3K5 and increased PGF and PGE secretion. Over-expression of p53 reversed the effect of ghrelin on OT, caused it to be inhibitory to P(4) secretion, but did not modify its action on MAP3K5, PGF, or PGE. FSH promoted the accumulation of p53, MAP3K5, and cyclin B1; these effects were unaffected by p53 transfection. These multiple effects of the p53 gene construct on luteinizing granulosa cells, cultured with and without hormones 1) demonstrate the effects of ghrelin and FSH on porcine ovarian cell apoptosis and secretory activity, 2) confirm the involvement of p53 in promoting apoptosis and inhibiting P(4) secretion in these cells, 3) provide the first evidence that p53 suppress proliferation of ovarian cells, 4) provide the first evidence that p53 is involved in the control of ovarian peptide hormone (OT) and prostaglandin (PGF and PGE) secretion, and 5) suggest that p53 can modulate, but probably not mediate, the effects of ghrelin and FSH on the ovary.

  5. YY1 positively regulates human UBIAD1 expression

    SciTech Connect

    Funahashi, Nobuaki; Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natumi; Watanabe, Masato; Suhara, Yoshitomo; Okano, Toshio

    2015-05-01

    Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K{sub 1}) and a series of bacterial menaquionones (MK-n; vitamin K{sub 2}). Menadione (vitamin K{sub 3}) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5′ rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter. - Highlights: • We cloned the human UBIAD1 promoter. • The functional importance of the YY1 motif was identified in the UBIAD1 promoter. • YY1 binds the UBIAD1 promoter in vitro and in vivo. • Knockdown of YY1 significantly decreased UBIAD1 expression. • YY1 up-regulates UBIAD1 conversion activity through the UBIAD1

  6. Characterization of Leukocyte Formin FMNL1 Expression in Human Tissues

    PubMed Central

    Heuser, Vanina D.; Iljin, Kristiina; Kampf, Caroline; Uhlen, Mathias; Carpén, Olli

    2014-01-01

    Formins are cytoskeleton regulating proteins characterized by a common FH2 structural domain. As key players in the assembly of actin filaments, formins direct dynamic cytoskeletal processes that influence cell shape, movement and adhesion. The large number of formin genes, fifteen in the human, suggests distinct tasks and expression patterns for individual family members, in addition to overlapping functions. Several formins have been associated with invasive cell properties in experimental models, linking them to cancer biology. One example is FMNL1, which is considered to be a leukocyte formin and is known to be overexpressed in lymphomas. Studies on FMNL1 and many other formins have been hampered by a lack of research tools, especially antibodies suitable for staining paraffin-embedded formalin-fixed tissues. Here we characterize, using bioinformatics tools and a validated antibody, the expression pattern of FMNL1 in human tissues and study its subcellular distribution. Our results indicate that FMNL1 expression is not restricted to hematopoietic tissues and that neoexpression of FMNL1 can be seen in epithelial cancer. PMID:24700756

  7. Flow cytometric analysis of glyoxalase-1 expression in human leukocytes.

    PubMed

    Skapare, Elina; Riekstina, Una; Liepinsh, Edgars; Konrade, Ilze; Makrecka, Marina; Maurina, Baiba; Dambrova, Maija

    2011-03-01

    Altered glyoxalase-1 (GLO-1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress- and aging-related diseases. In the present study, we developed a flow cytometry method for GLO-1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO-1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO-1 in leukocytes showed that expression index of GLO-1-positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO-1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO-1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress-related disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  8. Human lung expresses unique gamma-glutamyl transpeptidase transcripts.

    PubMed Central

    Wetmore, L A; Gerard, C; Drazen, J M

    1993-01-01

    gamma-Glutamyl transpeptidase (EC 2.3.2.2, gamma GT) is a membrane-bound ectoenzyme that plays an important role in the metabolism of glutathione. It is composed of two subunits, both of which are encoded by a common mRNA. We examined the expression of gamma GT in human lung tissue by Northern blot analysis and screening a cDNA library made from human lung poly(A)+ RNA. Our results show that there are two gamma GT mRNA populations in human lung tissue. We define these as group I (2.4 kb) and group II (approximately 1.2 kb) transcripts. In the present communication, we characterize the unique lung transcript. Sequence analysis of representative clones shows that group I transcripts are virtually identical to those previously isolated from liver and placenta but possess a unique 5' untranslated region. In marked contrast, group II transcripts appear to be human-lung-specific. Group II transcripts appear on Northern blots probed with full-length or 3'-biased gamma GT cDNA. Sequence analysis of group II clones shows them to be homologous with group I clones in the region that encodes the reading frame for the light chain; however, they possess a series of unique 5' untranslated regions, which suggests that they arise from lung-specific message processing. Additionally, approximately 50% of the isolated group II clones contain 34 nt substitutions compared with the "wild-type" gamma GT transcripts. These data indicate that human lung expresses unique gamma GT transcripts of unknown function as well as the classical form. The abundant group II transcripts may encode part of a heterodimer related to gamma GT or represent processed lung-specific pseudogenes. Images Fig. 1 PMID:7689219

  9. CCM2 expression during prenatal development and adult human neocortex.

    PubMed

    Tanriover, Gamze; Sozen, Berna; Gunel, Murat; Demir, Necdet

    2011-08-01

    Cerebral cavernous malformation (CCM) is one of the most common types of vascular malformations of the central nervous system, affecting nearly one in 200 people. CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium. Genetic linkage analyses have mapped three CCM loci to CCM1, CCM2 and CCM3. All three causative genes have now been identified allowing new insights into CCM pathophysiology. We focused on the CCM2 protein that might take place in blood vessel formation; we report here the expression patterns of CCM2 in prenatal development and adult human neocortex by means of immunohistochemistry and Western blot analysis. CCM2 was obviously detected in vascular endothelium and neuroglial precursor cells during development and also observed in arterial endothelium, neurons, some of the glial cells in adult neocortex. The expression patterns suggest that it could be one of the arterial markers whether this is a cause or a consequence of an altered vascular identity. CCM2 might play a role during vasculogenesis and angiogenesis during human brain development. Furthermore, with this study, CCM2 have been described for the first time in developing human neocortex.

  10. Cloning an expressed gene shared by the human sex chromosomes

    SciTech Connect

    Darling, S.M.; Banting, G.S.; Pym, B.; Wolfe, J.; Goodfellow, P.N.

    1986-01-01

    The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambdagt11 expression system in Escherichia coli and immunoscreening techniques, the authors have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. The authors conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.

  11. Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Pourhassan-Moghaddam, Mohammad; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space. PMID:27478780

  12. Reproductive Physiology in Young Men Is Cumulatively Affected by FSH-Action Modulating Genetic Variants: FSHR -29G/A and c.2039 A/G, FSHB -211G/T

    PubMed Central

    Grigorova, Marina; Punab, Margus; Punab, Anna Maria; Poolamets, Olev; Vihljajev, Vladimir; Žilaitienė, Birutė; Erenpreiss, Juris; Matulevičius, Valentinas; Laan, Maris

    2014-01-01

    Follicle-Stimulating Hormone Receptor (FSHR) -29G/A polymorphism (rs1394205) was reported to modulate gene expression and reproductive parameters in women, but data in men is limited. We aimed to bring evidence to the effect of FSHR -29G/A variants in men. In Baltic young male cohort (n = 982; Estonians, Latvians, Lithuanians; aged 20.2±2.0 years), the FSHR -29 A-allele was significantly associated with higher serum FSH (linear regression: effect 0.27 IU/L; P = 0.0019, resistant to Bonferroni correction for multiple testing) and showed a non-significant trend for association with higher LH (0.19 IU/L) and total testosterone (0.93 nmol/L), but reduced Inhibin B (−7.84 pg/mL) and total testes volume (effect −1.00 mL). Next, we extended the study and tested the effect of FSHR gene haplotypes determined by the allelic combination of FSHR -29G/A and a well-studied variant c.2039 A/G (Asn680Ser, exon 10). Among the FSHR -29A/2039G haplotype carriers (A-Ser; haplotype-based linear regression), this genetic effect was enhanced for FSH (effect 0.40 IU/L), Inhibin B (−16.57 pg/mL) and total testes volume (−2.34 mL). Finally, we estimated the total contribution of three known FSH-action modulating SNPs (FSHB -211G/T; FSHR -29G/A, c.2039 A/G) to phenotypic variance in reproductive parameters among young men. The major FSH-action modulating SNPs explained together 2.3%, 1.4%, 1.0 and 1.1% of the measured variance in serum FSH, Inhibin B, testosterone and total testes volume, respectively. In contrast to the young male cohort, neither FSHR -29G/A nor FSHR haplotypes appeared to systematically modulate the reproductive physiology of oligozoospermic idiopathic infertile patients (n = 641, Estonians; aged 31.5±6.0 years). In summary, this is the first study showing the significant effect of FSHR -29G/A on male serum FSH level. To account for the genetic effect of known common polymorphisms modulating FSH-action, we suggest haplotype-based analysis of FSHR SNPs

  13. Muscle Gene Expression Patterns in Human Rotator Cuff Pathology

    PubMed Central

    Choo, Alexander; McCarthy, Meagan; Pichika, Rajeswari; Sato, Eugene J.; Lieber, Richard L.; Schenk, Simon; Lane, John G.; Ward, Samuel R.

    2014-01-01

    Background: Rotator cuff pathology is a common source of shoulder pain with variable etiology and pathoanatomical characteristics. Pathological processes of fatty infiltration, muscle atrophy, and fibrosis have all been invoked as causes for poor outcomes after rotator cuff tear repair. The aims of this study were to measure the expression of key genes associated with adipogenesis, myogenesis, and fibrosis in human rotator cuff muscle after injury and to compare the expression among groups of patients with varied severities of rotator cuff pathology. Methods: Biopsies of the supraspinatus muscle were obtained arthroscopically from twenty-seven patients in the following operative groups: bursitis (n = 10), tendinopathy (n = 7), full-thickness rotator cuff tear (n = 8), and massive rotator cuff tear (n = 2). Quantitative polymerase chain reaction (qPCR) was performed to characterize gene expression pathways involved in myogenesis, adipogenesis, and fibrosis. Results: Patients with a massive tear demonstrated downregulation of the fibrogenic, adipogenic, and myogenic genes, indicating that the muscle was not in a state of active change and may have difficulty responding to stimuli. Patients with a full-thickness tear showed upregulation of fibrotic and adipogenic genes; at the tissue level, these correspond to the pathologies most detrimental to outcomes of surgical repair. Patients with bursitis or tendinopathy still expressed myogenic genes, indicating that the muscle may be attempting to accommodate the mechanical deficiencies induced by the tendon tear. Conclusions: Gene expression in human rotator cuff muscles varied according to tendon injury severity. Patients with bursitis and tendinopathy appeared to be expressing pro-myogenic genes, whereas patients with a full-thickness tear were expressing genes associated with fatty atrophy and fibrosis. In contrast, patients with a massive tear appeared to have downregulation of all gene programs except inhibition of

  14. Muscle gene expression patterns in human rotator cuff pathology.

    PubMed

    Choo, Alexander; McCarthy, Meagan; Pichika, Rajeswari; Sato, Eugene J; Lieber, Richard L; Schenk, Simon; Lane, John G; Ward, Samuel R

    2014-09-17

    Rotator cuff pathology is a common source of shoulder pain with variable etiology and pathoanatomical characteristics. Pathological processes of fatty infiltration, muscle atrophy, and fibrosis have all been invoked as causes for poor outcomes after rotator cuff tear repair. The aims of this study were to measure the expression of key genes associated with adipogenesis, myogenesis, and fibrosis in human rotator cuff muscle after injury and to compare the expression among groups of patients with varied severities of rotator cuff pathology. Biopsies of the supraspinatus muscle were obtained arthroscopically from twenty-seven patients in the following operative groups: bursitis (n = 10), tendinopathy (n = 7), full-thickness rotator cuff tear (n = 8), and massive rotator cuff tear (n = 2). Quantitative polymerase chain reaction (qPCR) was performed to characterize gene expression pathways involved in myogenesis, adipogenesis, and fibrosis. Patients with a massive tear demonstrated downregulation of the fibrogenic, adipogenic, and myogenic genes, indicating that the muscle was not in a state of active change and may have difficulty responding to stimuli. Patients with a full-thickness tear showed upregulation of fibrotic and adipogenic genes; at the tissue level, these correspond to the pathologies most detrimental to outcomes of surgical repair. Patients with bursitis or tendinopathy still expressed myogenic genes, indicating that the muscle may be attempting to accommodate the mechanical deficiencies induced by the tendon tear. Gene expression in human rotator cuff muscles varied according to tendon injury severity. Patients with bursitis and tendinopathy appeared to be expressing pro-myogenic genes, whereas patients with a full-thickness tear were expressing genes associated with fatty atrophy and fibrosis. In contrast, patients with a massive tear appeared to have downregulation of all gene programs except inhibition of myogenesis. These data highlight the

  15. Harpagoside suppresses IL-6 expression in primary human osteoarthritis chondrocytes.

    PubMed

    Haseeb, Abdul; Ansari, Mohammad Yunus; Haqqi, Tariq M

    2017-02-01

    There is growing evidence in support of the involvement of inflammatory response in the pathogenesis of osteoarthritis (OA). Harpagoside, one of the bioactive components of Harpagophytum procumbens (Hp), has been shown to possess anti-inflammatory properties. Here we used an in vitro model of inflammation in OA to investigate the potential of harpagoside to suppress the production of inflammatory cytokines/chemokines such as IL-6 and matrix degrading proteases. We further investigated the likely targets of harpagoside in primary human OA chondrocytes. OA chondrocytes were pre-treated with harpagoside before stimulation with IL-1β. mRNA expression profile of 92 cytokines/chemokines was determined using TaqMan Human Chemokine PCR Array. Expression levels of selected mRNAs were confirmed using TaqMan assays. Protein levels of IL-6 and MMP-13 were assayed by ELISA and immunoblotting. Total protein levels and phosphorylation of signaling proteins were determined by immunoblotting. Cellular localization of IL-6 and c-Fos was performed by immunofluorescence and confocal microscopy. DNA binding activity of c-FOS/AP-1 was determined by ELISA. Harpagoside significantly altered the global chemokine expression profile in IL-1β-stimulated OA chondrocytes. Expression of IL-6 was highly induced by IL-1β, which was significantly inhibited by pre-treatment of OA chondrocytes with harpagoside. Harpagoside did not inhibit the IL-1β-induced activation of NF-κB and C/EBPβ transcription factors but suppressed the IL-1β-triggered induction, phosphorylation, and DNA binding activity of c-FOS, one of the main components of AP-1 transcription factors. Further, harpagoside significantly inhibited the expression of MMP-13 in OA chondrocytes under pathological conditions. siRNA-mediated knockdown of IL-6 resulted in suppressed expression and secretion of MMP-13 directly linking the role of IL-6 with MMP-13 expression. Taken together, the present study suggests that harpagoside exerts a

  16. Developmental gene expression profiles of the human pathogen Schistosoma japonicum

    PubMed Central

    Gobert, Geoffrey N; Moertel, Luke; Brindley, Paul J; McManus, Donald P

    2009-01-01

    Background The schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle – aquatic/snail (eggs, miracidia, sporocysts, cercariae), juvenile (lung schistosomula and paired but pre-egg laying adults) and adult (paired, mature males and egg-producing females, both examined separately). Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite. Results Gene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence. Conclusion The findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis. PMID:19320991

  17. Epigenetic regulation of CIITA expression in human T-cells.

    PubMed

    van Eggermond, Marja C J A; Boom, Daniël R; Klous, Petra; Schooten, Erik; Marquez, Victor E; Wierda, Rutger J; Holling, Tjadine M; van den Elsen, Peter J

    2011-11-15

    In humans, T-cells accomplish expression of MHC-II molecules through induction of CIITA upon activation. Here we show that CIITA promoter accessibility in T-cells is epigenetically regulated. In unstimulated T-cells, CIITA-PIII chromatin displays relative high levels of repressive histone methylation marks (3Me-K27-H3 and 3Me-K20-H4) and low levels of acetylated histones H3 (Ac-H3) and H4 (Ac-H4). These repressive histone marks are replaced by histone methylation marks associated with transcriptional active genes (3Me-K4-H3) and high levels of Ac-H3 and Ac-H4 in activated T-cells. This is associated with concomitant recruitment of RNA polymerase II. In T-leukemia cells, devoid of CIITA expression, similar repressive histone methylation marks and low levels of acetylated histone H3 correlated with lack of CIITA expression. This in contrast to CIITA expressing T-lymphoma cells, which display high levels of Ac-H3 and 3Me-K4-H3, and relative low levels of the 3Me-K27-H3 and 3Me-K20-H4 marks. Of interest was the observation that the levels of histone acetylation and methylation modifications in histones H3 and H4 were also noted in chromatin of the downstream CIITA-PIV promoter as well as the upstream CIITA-PI and CIITA-PII promoters both in normal T-cells and in malignant T-cells. Together our data show that CIITA chromatin in T-cells expressing CIITA display similar histone acetylation and methylation characteristics associated with an open chromatin structure. The opposite is true for T-cells lacking CIITA expression, which display histone modifications characteristic of condensed chromatin. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Sexual Dimorphism in Clock Genes Expression in Human Adipose Tissue

    PubMed Central

    Gómez-Abellán, P.; Madrid, J. A.; Luján, J. A.; Frutos, M. D.; González, R.; Martínez-Augustín, O.; de Medina, F. Sánchez; Ordovás, J. M.; Garaulet, M.

    2015-01-01

    Background This study was carried out to investigate whether sex-related differences exist in the adipocyte expression of clock genes from subcutaneous abdominal and visceral fat depots in severely obese patients. Methods We investigated 16 morbidly obese patients, eight men and eight women (mean age 45±20 years; mean BMI 46±6 kg/m2), undergoing laparoscopic gastric bypass surgery. Biopsies were taken as paired samples [subcutaneous and visceral adipose tissue (AT)] at the beginning of the surgical process at 11:00 h in the morning. Metabolic syndrome features such as waist circumference, plasma glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also studied. The expression of clock genes (PER2, BMAL1, and CRY1) was measured by quantitative real-time PCR, Western blot, and immunohistochemical analysis. Results Gene expression was significantly higher in women than in men for the three genes studied in both ATs (P<0.05). In visceral fat, these differences were more marked. (P<0.001). Western blot analysis partially confirmed these results since statistical differences were observed for PER2 in both ATs and for CRY1 in subcutaneous adipose tissue. There were no differences in BMAL1 protein expression. Interestingly, clock gene expression level was correlated with LDL-C and HDL-C (P<0.05). Moreover, we found significant associations with body fat mass in women and with age in men. Conclusions Clock genes expression is sex dependent in human adipose tissue from morbidly obese subjects and correlates to a decreased in metabolic syndrome-related traits. These preliminary results make necessary to go deep into the knowledge of the molecular basis of the sexual dimorphism in chronobiology. PMID:22081238

  19. LIN28A Expression Reduces Sickling of Cultured Human Erythrocytes

    PubMed Central

    de Vasconcellos, Jaira F.; Fasano, Ross M.; Lee, Y. Terry; Kaushal, Megha; Byrnes, Colleen; Meier, Emily R.; Anderson, Molly; Rabel, Antoinette; Braylan, Raul; Stroncek, David F.; Miller, Jeffery L.

    2014-01-01

    Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes. PMID:25188417

  20. RORα and RORγ expression inversely correlates with human melanoma progression

    PubMed Central

    Brożyna, Anna A.; Jóźwicki, Wojciech; Skobowiat, Cezary; Jetten, Anton; Slominski, Andrzej T.

    2016-01-01

    The retinoic acid-related orphan receptors (RORs) regulate several physiological and pathological processes, including immune functions, development and cancer. To study the potential role of RORs in melanoma progression, we analysed RORα and RORγ expression in nevi and primary melanomas and non-lesional skin and metastases in relation to melanoma clinico-pathomorphological features. The expression of RORα and RORγ was lower in melanomas than in nevi and decreased during melanoma progression, with lowest levels found in primary melanomas at stages III and IV and in melanoma metastases. Their expression correlated with pathomorphological pTNM parameters being low in aggressive tumors and being high in tumors showing histological markers of good prognosis. Higher nuclear levels of RORα and RORγ and of cytoplasmic RORγ correlated with significantly longer overall and disease free survival time. Highly pigmented melanomas showed significantly lower level of nuclear RORs. This study shows that human melanoma development and aggressiveness is associated with decreased expression of RORα and RORγ, suggesting that RORs could be important in melanoma progression and host responses against the tumor. Furthermore, it suggests that RORα and RORγ might constitute a novel druggable target in anti-melanoma management using tumor suppressor gene therapy restoring their normal functions. PMID:27542227

  1. Expression of Formyl-peptide Receptors in Human Lung Carcinoma.

    PubMed

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; Lucariello, Angela; De Luca, Antonio; De Rosa, Nicolina; Mazzarella, Gennaro; Bianco, Andrea; Ammendola, Rosario

    2015-05-01

    Formyl-peptide receptors (FPRs) are expressed in several tissues and cell types. The identification of markers involved in cell growth may further allow for molecular profiling of lung cancer. We investigated the possible role of FPRs as molecular markers in several types of lung carcinomas which is the main cause of cancer death worldwide. Tumor tissue samples were collected from six patients affected by lung cancer. Biopsies were analyzed for expression of FPR isoforms both in tumoral and peritumoral tissue by real-time polymerase chain reaction (PCR), western blot and immunofluorescence. Real-time PCR, western blot and immunofluorescence analyses showed that FPR expression is lower in types of human lung cancer tissues when compared to the surrounding peritumoral tissues. The study of the mechanistic basis for the control of FPR expression in normal peritumoral versus tumoral tissues could provide the basis for new diagnostic and therapeutic interventions. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. Expression of human peripheral cannabinoid receptor for structural studies

    PubMed Central

    Yeliseev, Alexei A.; Wong, Karen K.; Soubias, Olivier; Gawrisch, Klaus

    2005-01-01

    Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%–90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques. PMID:16195551

  3. Bradykinin promotes TLR2 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio

    2011-12-01

    Bradykinin (BK) is implicated in the sensation of pain, vasodilation, increases in vascular permeability and pathogenic processes associated with inflammation. Studies have shown that BK promotes the intracellular movement of calcium in human gingival fibroblasts by binding to the B2 receptor. In this study we investigated the effect of BK on regulation of Toll-like receptor 2 (TLR2) expression. Our results show that BK stimulates TLR2 receptor transcription and translation by activation of protein kinase C as well as AKT. Our study contributes important information on the regulation and expression of molecules that promote chronic inflammatory processes, which lead to periodontitis and consequently to loss of the dental organ. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Human HLA-Ev (147) Expression in Transgenic Animals.

    PubMed

    Matsuura, R; Maeda, A; Sakai, R; Eguchi, H; Lo, P-C; Hasuwa, H; Ikawa, M; Nakahata, K; Zenitani, M; Yamamichi, T; Umeda, S; Deguchi, K; Okuyama, H; Miyagawa, S

    2016-05-01

    In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice. A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method. The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung. A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Establishment a CHO Cell Line Expressing Human CD52 Molecule

    PubMed Central

    Kadijeh, Tati; Mahsa, Yazdanpanah-Samani; Amin, Ramezani; Elham, Mahmoudi Maymand; Abbas, Ghaderi

    2016-01-01

    Background: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details Methods: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. Results: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. Conclusion: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protei