227. FSH receptor expression in small human ovarian follicles

2005 ◽  
Vol 17 (9) ◽  
pp. 88
Author(s):  
J. H. Quennell ◽  
J. L. Stanton ◽  
P. R. Hurst
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Real Time Pcr ◽  
In Situ Hybridisation ◽  
Receptor Expression ◽  
Follicle Development ◽  
Fsh Receptor ◽  
Relative Expression ◽  
Receptor Mrna

Follicle-stimulating hormone (FSH) is pivotal in ovarian follicle development; the granulosa cells are the targets of FSH action in the ovary via FSH receptors. Granulosa cell growth and division mark initial follicle recruitment. The acquisition of FSH receptors on granulosa cells is regarded as a key event in hormone responsiveness and consequently follicle development. Due to the low abundance of FSH receptors and low expression of its mRNA it has been difficult to definitively characterise FSH receptor expression patterns. Here, localisation of FSH receptor in different follicle populations has been assessed with in situ hybridisation and real-time PCR of laser microdissected samples. We have used non-radioactive in situ hybridisation to investigate FSH receptor mRNA on a wide range of follicle stages. Biopsies from healthy fertile women (28–33 years) were frozen, embedded and cryosectioned at 10 µm. DIG-labelled RNA probes were designed to detect all splice variants. Hybridised probes were detected with NBT/BCIP in a colorimetric reaction. Secondly, follicles of different morphometric stages were isolated with a laser microscope. RNA extraction, reverse transcription and real-time PCR were used to confirm RNA presence and quantify relative expression. All follicle stages (from primordial to large antral) showed the presence of FSH receptor mRNA in their granulosa cells; sense controls were negative. Observations from real-time PCR indicate FSH receptor mRNA is present in all follicle stages observed and relative expression levels increase over early follicle development. These results challenge the existing doctrine that FSH receptor is absent in the smallest follicles. This suggests initial follicle recruitment may involve gonadotrophins. The use of sensitive molecular techniques will be crucial in elucidating this further.

scholarly journals Fluorophores from fungi

2003 ◽  
Vol 24 (3) ◽  
pp. 12 ◽  
Author(s):  
Duncan Veal ◽  
Philip Bell ◽  
Hayley Brown ◽  
Hung-Yoon Choi ◽  
Peter Karuso
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Microarray ◽  
Differential Display ◽  
Real Time Pcr ◽  
In Situ Hybridisation ◽  
Analytical Techniques ◽  
Fluorescence In Situ Hybridisation

Fluorescence has many advantages over traditional colour and radioactive labels, and is playing an increasingly important role in the most powerful analytical techniques. For example, fluorescence is at the heart of many nucleic acid based diagnostics (e.g. DNA microarray, real time-PCR, fluorescence in situ hybridisation, etc), immunofluorescence assays, defined substrate technologies and differential display proteomics and is gradually replacing or complementing other techniques based on colour or radiolabels.

Association of MicroRNA Expression Profiles with Karyotype in Acute Myeloid Leukaemias Revealed by Real-Time PCR and In Situ Hybridisation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 105-105
Author(s):  
Amanda Dixon-McIver ◽  
Phil East ◽  
Charles A. Mein ◽  
Jean-Baptiste Cazier ◽  
Gael Molloy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
In Situ Hybridisation ◽  
Locked Nucleic Acid ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract MicroRNAs (miRNAs) are single stranded non-coding RNAs of ∼ 22 nucleotides involved in gene regulation. Several examples of an association between disrupted expression of miRNAs and cancer have been shown. Using a real-time quantitative PCR, designed to amplify only from the mature miRNA (TaqMan® -MicroRNA Looped-PCR assay, Applied Biosystems), we measured the expression levels of 157 miRNAs in 100 acute myeloid leukaemia (AML) patients representing the spectrum of known karyotypes common in AML, 2 leukaemic cell lines (KG1 and NB4), and the bone marrow from 2 healthy donors. ANOVA analysis using a 5% false discovery rate threshold was performed to identify differentially expressed miRNAs between leukaemia samples and normal bone marrow. MiRNAs specific to karyotype groupings were also identified in the same way. A method was developed to demonstrate the spatial localisation, in situ, of specific miRNA identified in the quantification and confirm the expression of miRNA with relation to karyotype. Commercial locked nucleic acid (LNA) oligonucleotides (Exiqon) were obtained for two miRNAs (miRNA-127 and miRNA-154), as well as for positive and negative control probes. LNAs were labelled with digoxigenin and probes applied to both cytospins and/or trephines of a sample set representative of the different AML subtypes. Detection of hybridisation signals was either by colorimetric or fluorescent reaction and visualised by confocal microscopy. Unsupervised cluster analysis revealed an association of miRNA expression with the karyotype of the samples. Promyelocytic leukaemias (APML) bearing the t(15;17) translocation, show a distinct pattern characterised by the high expression of a subset of 10 miRNAs located in the human 14q32 imprinted domain, including miR-127 and miR-154. ANOVA data analysis revealed the de-regulation of 33 miRNAs across the leukaemic set in respect to bone marrow from healthy donors. Seventeen miRNAs were up-regulated and 16 down-regulated. MiRNAs miR-155, miR-181a, miR-181b, miR-181c, miR-142-5p, miR-221, and miR-222, were among those commonly highly expressed. Down-regulated were miR-26a, miR-34c, and miR-199a. MiRNAs miR-10a and miR-125b showed the highest variability throughout the samples, being associated with specific subgroups. In situ hybridisation analysis of miR-127 and miR-154 confirmed the results obtained by real-time PCR of their expression associated with APML. This study, conducted on about a third of the miRNAs reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer. The expression analysis of larger number of miRNAs coupled with the in situ hybridisation of leukaemic cells will allow the investigation of miRNA expression on stored samples during the disease course and provide valuable insights into the leukaemogenic process.

Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cyclic AMP and activin

1997 ◽  
Vol 153 (3) ◽  
pp. 465-473 ◽  
Author(s):  
M Tano ◽  
T Minegishi ◽  
K Nakamura ◽  
S Karino ◽  
Y Ibuki ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Receptor Expression ◽  
Northern Blot Hybridization ◽  
Fsh Receptor ◽  
Mrna Levels ◽  
Mrna Transcript ◽  
Receptor Mrna ◽  
Degradation Rates ◽  
Mrna Transcripts ◽  
In Cells

Abstract The effect of FSH on the induction of FSH receptors in granulosa cells is believed to be mediated, at least in part, by the cAMP second messenger system. We examined the effect of activin and cAMP on FSH receptor expression in this culture system. Steady-state levels of FSH receptor mRNA, analyzed by Northern blot hybridization, increased 3·5-fold in response to 24-h incubation with activin and 1·7-fold with 12-h incubation with 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0·2 mm). We have investigated whether 8-Br-cAMP- and/or activin-induced increases in FSH receptor mRNA levels are the result of increased transcription and/or altered mRNA stability. The rates of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, increased 3-fold in cells treated with activin and 1·5-fold in cells treated with 8-Br-cAMP for 2 h. To examine the degradation rates of FSH receptor mRNA transcripts, granulosa cells were preincubated with 8-Br-cAMP, activin, or medium alone for 6 h. After the preincubation period, 5 μm actinomycin-D or 200 μm 5,6-dichloro-1-β-ribofuranosyl benzimidazole were added to arrest new RNA synthesis. The decay curves for the 2·4 kb FSH receptor mRNA transcript in granulosa cells were not significantly different in the absence or presence of 8-Br-cAMP. Activin, on the other hand, significantly altered the slope of the FSH receptor mRNA decay curve and increased the half-life of the 2·4 kb FSH receptor mRNA transcript. These data provide evidence that cAMP induces FSH receptor mRNA levels by stimulating the transcription rate and that activin increases FSH receptor mRNA levels both by stimulating transcription rates and by stabilizing the FSH receptor mRNA transcripts. Journal of Endocrinology (1997) 153, 465–473

Development of a real-time PCR assay for the detection of Trichinella spiralis in situ

2009 ◽  
Vol 161 (1-2) ◽  
pp. 92-98 ◽  
Author(s):  
H. Atterby ◽  
J. Learmount ◽  
C. Conyers ◽  
I. Zimmer ◽  
N. Boonham ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Trichinella Spiralis ◽  
Pcr Assay

scholarly journals Comparison of the sensitivity and specificity of real-time PCR and in situ hybridization in HPV16 and 18 detection in archival cervical cancer specimens

2012 ◽  
Vol 50 (2) ◽  
pp. 239-247 ◽  
Author(s):  
Beata Biesaga ◽  
Sława Szostek ◽  
Małgorzata Klimek ◽  
Jerzy Jakubowicz ◽  
Joanna Wysocka
Keyword(s):  
Cervical Cancer ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr

128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

2016 ◽  
Vol 28 (2) ◽  
pp. 194 ◽  
Author(s):  
L. F. Schütz ◽  
J. E. Ervin ◽  
L. Zhang ◽  
C. Robinson ◽  
M. Totty ◽  
...  
Keyword(s):  
Growth Factor ◽  
Real Time ◽  
Granulosa Cells ◽  
Real Time Pcr ◽  
Linear Models ◽  
Rna Extraction ◽  
Insulin Like Growth Factor ◽  
Steroid Production ◽  
Oestradiol Production

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.

209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 252
Author(s):  
G. K. Deb ◽  
S. R. Dey ◽  
K. S. Huque ◽  
M. Fokruzzaman ◽  
K. L. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Implantation ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Receptor Expression ◽  
Bovine Embryo

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.

Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real-time PCR

2009 ◽  
Vol 2 (1) ◽  
pp. 76-80 ◽  
Author(s):  
Craig Baker-Austin ◽  
Anthony Gore ◽  
James D. Oliver ◽  
Rachel Rangdale ◽  
J Vaun McArthur ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
In Situ Detection
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