70 EXPRESSION OF NUCLEAR AND MEMBRANE PROGESTERONE RECEPTORS IN THE CANINE OVIDUCT DURING THE PERIOVULATORY PERIOD

2012 ◽  
Vol 24 (1) ◽  
pp. 147 ◽  
Author(s):  
M. Z. Tahir ◽  
K. Reynaud ◽  
S. Thoumire ◽  
S. Chastant-Maillard ◽  
M. Saint-Dizier
Keyword(s):  
Target Genes ◽  
Geometric Mean ◽  
Progesterone Receptors ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Standard Curve ◽  
Relative Standard ◽  
Relative Standard Curve Method ◽  
Curve Method ◽  
Roche Diagnostics

In the bitch, the oviduct is the site of oocyte maturation (Day 1 to 3 after ovulation), sperm transport/capacitation, fertilization (Day 3 to 4) and embryo development to the morula/blastocyst stage (Day 4 to 8). Unlike other mammals, these events occur in the presence of high (>6 ng mL–1) and increasing plasma levels of progesterone (P4), but little is known about the regulation of oviductal functions by P4 in the bitch. The objective of this work was to study the mRNA expression of nuclear (PR) and membrane (PGRMC1, PGRMC2, mPRβ and mPRγ) P4 receptors in the canine oviduct during the periovulatory period. Thirty-six Beagle bitches were ovariectomized at 6 stages: anestrus, before the LH peak (pre-LH), after the LH peak (pre-ov) and after ovulation (Day 1, 4 and 7). Three oviductal regions were collected [i.e. ampulla, isthmus and utero-tubal junction (UTJ)]. Total RNA was extracted and then reverse transcribed. The expression of target genes was assessed in duplicate by quantitative PCR (LightCycler® 480; Roche Diagnostics, Meylan, France) using the relative standard curve method and normalized by the geometric mean of 2 reference genes (RPS19 and GAPDH). Relative amounts of mRNA were compared between groups by ANOVA followed, when necessary, by Duncan's test. The expression of nuclear and membrane P4 receptor mRNA varied according to the stage. Expression of PR mRNA was significantly higher at pre-LH, pre-ov and Day 1 stages [means of 1.8, 1.6 and 1.5 arbitrary units (AU), respectively] than at anoestrus, Day 4 and Day 7 (1, 0.4 and 0.5 AU, respectively) in the ampulla. Same patterns of expression were observed for PR in the isthmus and UTJ. Expression of PGRMC1 and PGRMC2 mRNA were at the lowest level during anoestrus (1 AU) and increased significantly from pre-LH to Day 7 in the ampulla (from 2.2 to 8.3 AU and from 1.3 to 5.4 AU for PGRMC1 and PGRMC2, respectively) and in the isthmus (from 0.4 to 2.6 AU and from 0.5 to 1.8 AU for PGRMC1 and PGRMC2, respectively). In the UTJ, mRNA levels for PGRMC1 and PGRMC2 were the highest at Day 4 (3.9 AU) and pre-LH (2.1 AU), respectively, compared to other stages. Expression of mPRβ mRNA did not vary according to the stage in the ampulla and the isthmus, whereas it was significantly lower at Days 4 and 7 (0.6–0.7 AU) compared to other stages (1–1.2 AU) in the UTJ. Expression of mPRγ was significantly higher at Day 7 (5.0 AU) compared to other stages (0.2–1 AU) in the ampulla and was significantly higher at both anoestrus (1 AU) and Day 7 (0.9 AU) compared to other stages (0.02–0.09 AU) in the isthmus, whereas it did not vary significantly in the UTJ. In conclusion, our data suggests that P4 may be an important regulating factor of oviductal functions and could mediate its actions through genomic as well as non-genomic pathways.

121 EXPRESSION OF STEROID RECEPTORS IN THE CUMULUS - OOCYTE COMPLEX AROUND OVULATION IN THE BITCH

2014 ◽  
Vol 26 (1) ◽  
pp. 174 ◽  
Author(s):  
M. Z. Tahir ◽  
K. Reynaud ◽  
S. Thoumire ◽  
S. Chastant-Maillard ◽  
M. Saint-Dizier
Keyword(s):  
Oocyte Maturation ◽  
Steroid Receptors ◽  
Geometric Mean ◽  
Plasma Concentrations ◽  
Cumulus Cells ◽  
Immature Stage ◽  
Mean Value ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Relative Standard

In the bitch, oocytes are ovulated at an immature stage (prophase I) and resume meiosis in the oviduct, 3 to 4 days after ovulation while they are still surrounded by 2 to 3 layers of cumulus cells. Canine cumulus-oocyte complexes (COC) are exposed to high and changing plasma concentrations of 17β-oestradiol (E2) and progesterone (P4) during the periovulatory period. In order to explore whether oocyte maturation may be regulated by steroids in this species, the expression of E2 (ERα, ERβ) and P4 (nuclear: PR; membrane: PGRMC1, PGRMC2, mPRα, mPRβ, mPRγ) receptors was studied in COC at precise times around ovulation. Ovaries were collected from Beagle bitches during anestrus (n = 4), after the beginning of proestrus, and before the LH peak (Pre-LH, n = 7), after the LH peak and before ovulation (Pre-ov, n = 8), and at Day 1 (n = 11) and Day 4 (n = 8) post-ovulation. Anoestrus COC were recovered from follicles smaller than 1 mm in diameter. The COC at the Pre-LH and Pre-ov stages were aspirated from preovulatory follicles (4.5–6 mm in diameter). Such follicular COC were partially denuded to leave the 2 to 3 innermost cumulus layers firmly attached to the zona pellucida. Post-ovulatory COC, naturally surrounded by 2 to 3 cumulus layers, were recovered by oviductal flushing. Total RNA was extracted from 3 batches of 10 COC per stage, then reverse transcribed. The expression of steroid receptors was assessed in duplicate by qPCR (LightCycler® 480, Roche Diagnostics) using the relative standard curve method and normalized by the geometric mean value of the two most stable reference genes (BGLR and RPS5; NormFinder software) chosen among four genes previously tested. Relative amounts of mRNA levels were compared between stages by ANOVA followed, when necessary, by a Tukey test. The ERα and ERβ expression did not vary significantly with the stage. In contrast, a significant variation between stages in nuclear and 4 membrane P4 receptor expression was observed (P < 0.0001 for PR; P < 0.001 for PGRMC1 and mPRβ; P < 0.05 for PGRMC2 and mPRγ). The PR mRNA levels were significantly higher at Pre-ov than at any other stage. PGRMC1 expression was significantly higher at Pre-ov and Day 4 compared with anestrus and Pre-LH, and was at an intermediate level at Day 1. The expression of PGRMC2, mPRβ, and mPRγ remained low from anestrus to Day 1 and increased significantly at Day 4. Lastly, mRNA levels of mPRα were below the detection limit at all stages. This is the first report of steroid receptor expression in canine COC at precise times around ovulation. The stage-specific variation in expression of nuclear and of several membrane P4 receptors around ovulation suggests a role for P4 in canine oocyte maturation. The exact localisation of these receptors in cumulus cells, oocytes, or both remains to be determined.

Atomic Absorption Determination of Nickel in Heavy Crudes via Carbon Rod Atomizer

1977 ◽  
Vol 31 (3) ◽  
pp. 207-210 ◽  
Author(s):  
J. J. Labrecque ◽  
J. Galobardes ◽  
M. E. Cohen
Keyword(s):  
Atomic Absorption ◽  
Standard Curve ◽  
Determination Of Nickel ◽  
Absorption Determination ◽  
Relative Standard ◽  
Curve Method ◽  
Standard Additions ◽  
Standard Curve Method ◽  
Carbon Rod Atomizer

A method is described for the determination of nickel in heavy crude oil by carbon rod atomic absorption spectroscopy in the parts per million range. A 5-g sample is completely dissolved to 25 ml of tetrahydrofuran (THF) stabilized with 0.1% hydroquinone and further diluted with THF. Nickel can be measured at a concentration of 0.1 ppm with a relative standard deviation of about 10% after a 100-fold dilution of the crude. The analysis by standard curve method as well as standard additions methods gave reproducible results. These results also agreed well with values obtained by conventional activation analysis. The method is relatively rapid after sample dilution.

A Comparative Analysis of Methods for Titering Reovirus

2020 ◽  
Vol 8 (5) ◽  
pp. 409-417
Author(s):  
Yi-Chen Yang ◽  
Xian-Yao Wang ◽  
Yuan-Yuan An ◽  
Chun-Xiang Liao ◽  
Nian-Xue Wang ◽  
...  
Keyword(s):  
Traditional Method ◽  
Virus Titer ◽  
Cell Analysis ◽  
Half Cell ◽  
L929 Cells ◽  
Cell Index ◽  
Standard Curve ◽  
Analysis Technique ◽  
Curve Method ◽  
Standard Curve Method

Background: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. Objective: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT50) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID50) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology. Methods: : Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI50) values of reovirus infected L929 cells with TCID50 titers were analyzed by RTCA, the CI50-TCID50 standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT50-TCID50 standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID50 method. Results: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL. Conclusion: The CIT50-TCID50 standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID50 method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID50 method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

scholarly journals Extract of Wax Gourd Peel Prevents High-Fat Diet-Induced Hyperlipidemia in C57BL/6 Mice via the Inhibition of the PPARγPathway

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Ming Gu ◽  
Shengjie Fan ◽  
Gaigai Liu ◽  
Lu Guo ◽  
Xiaobo Ding ◽  
...  
Keyword(s):  
Blood Glucose ◽  
High Fat Diet ◽  
Target Genes ◽  
Metabolic Diseases ◽  
Body Weight Gain ◽  
Fasting Blood Glucose ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
High Fat ◽  
Wax Gourd

Wax gourd is a popular vegetable in East Asia. In traditional Chinese medicine, wax gourd peel is used to prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, and cardiovascular disease. However, there is no experimental evidence to support these applications. Here, we examined the effect of the extract of wax gourd peel (EWGP) on metabolic disorders in diet-induced C57BL/6 obese mice. In the preventive experiment, EWGP blocked body weight gain and lowered serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), liver TG and TC contents, and fasting blood glucose in mice fed with a high-fat diet. In the therapeutic study, we induced obesity in the mice and treated with EWGP for two weeks. We found that EWGP treatment reduced serum and liver triglyceride (TG) contents and fasting blood glucose and improved glucose tolerance in the mice. Reporter assay and gene expression analysis showed that EWGP could inhibit peroxisome proliferator-activated receptorγ(PPARγ) transactivities and could decrease mRNA levels of PPARγand its target genes. We also found that HMG-CoA reductase (HMGCR) was downregulated in the mouse liver by EWGP. Our data suggest that EWGP lowers hyperlipidemia of C57BL/6 mice induced by high-fat diet via the inhibition of PPARγand HMGCR signaling.

scholarly journals Conditional Deletion of the Retinoblastoma (Rb) Gene in Ovarian Granulosa Cells Leads to Premature Ovarian Failure

2008 ◽  
Vol 22 (9) ◽  
pp. 2141-2161 ◽  
Author(s):  
Claudia Andreu-Vieyra ◽  
Ruihong Chen ◽  
Martin M. Matzuk
Keyword(s):  
Granulosa Cell ◽  
Target Genes ◽  
Ovarian Function ◽  
Specific Pattern ◽  
Tumor Formation ◽  
Conditional Knockout ◽  
Mrna Levels ◽  
Preantral Follicles ◽  
Recombination System ◽  
Ovarian Tumorigenesis

Abstract The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.

scholarly journals Simultaneous Determination of Nitroimidazoles and Quinolones in Honey by Modified QuEChERS and LC-MS/MS Analysis

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Haiyan Lei ◽  
Jianbo Guo ◽  
Zhuo Lv ◽  
Xiaohong Zhu ◽  
Xiaofeng Xue ◽  
...  
Keyword(s):  
Recovery Rates ◽  
Inhibition Effect ◽  
Standard Curve ◽  
Modified Quechers ◽  
Relative Standard ◽  
Acacia Honey ◽  
The Matrix ◽  
Liquid Chromatography Tandem Mass ◽  
Standard Deviations

This study reports an analytical method for the determination of nitroimidazole and quinolones in honey using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A modified QuEChERS methodology was used to extract the analytes and determine veterinary drugs in honey by LC-MS/MS. The linear regression was excellent at the concentration levels of 1–100 ng/mL in the solution standard curve and the matrix standard curve. The recovery rates of nitroimidazole and quinolones were 4.4% to 59.1% and 9.8% to 46.2% with relative standard deviations (RSDs) below 5.2% and the recovery rates of nitroimidazole and quinolones by the matrix standard curve ranged from 82.0% to 117.8% and 79% to 115.9% with relative standard deviations (RSDs) lower than 6.3% in acacia and jujube honey. The acacia and jujube honeys have stronger matrix inhibition effect to nitroimidazole and quinolones residue; the matrix inhibition effect of jujube honey is stronger than acacia honey. The matrix standard curve can calibrate matrix effect effectively. In this study, the detection method of antibiotics in honey can be applied to the actual sample. The results demonstrated that the modified QuEChERS method combined with LC-MS/MS is a rapid, high, sensitive method for the analysis of nitroimidazoles and quinolones residues in honey.

scholarly journals Birdsong Decreases Protein Levels of FoxP2, a Molecule Required for Human Speech

2008 ◽  
Vol 100 (4) ◽  
pp. 2015-2025 ◽  
Author(s):  
Julie E. Miller ◽  
Elizabeth Spiteri ◽  
Michael C. Condro ◽  
Ryan T. Dosumu-Johnson ◽  
Daniel H. Geschwind ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Target Genes ◽  
Expression Patterns ◽  
Vocal Learning ◽  
Brain Regions ◽  
Motor Deficits ◽  
Mrna Levels ◽  
Adult Males ◽  
Protein Levels ◽  
Area X

Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability.

scholarly journals The Role of the FOXO1/β2-AR/p-NF-κB p65 Pathway in the Development of Endometrial Stromal Cells in Pregnant Mice under Restraint Stress

2021 ◽  
Vol 22 (3) ◽  
pp. 1478
Author(s):  
Jiayin Lu ◽  
Yaoxing Chen ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong
Keyword(s):  
Oxidative Stress ◽  
Stromal Cells ◽  
Restraint Stress ◽  
Target Genes ◽  
Mrna Levels ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Pregnant Mice

Restraint stress causes various maternal diseases during pregnancy. β2-Adrenergic receptor (β2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the β2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the β2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, β2-AR, and even the protein levels of FOXO1 and β2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when β2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the β2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.

Stress Hormone System and Epigenetics in Depression

2017 ◽  
Vol 41 (S1) ◽  
pp. S19-S19
Author(s):  
V. O’Keane ◽  
C. Farrell ◽  
K. Doolin ◽  
J. Chai ◽  
N. O’leary ◽  
...  
Keyword(s):  
Hpa Axis ◽  
Target Genes ◽  
Receptor Gene ◽  
Cell Receptor ◽  
Mrna Levels ◽  
Early Life Adversity ◽  
Generational Transmission ◽  
T Cell Receptor Gene ◽  
Competing Interest ◽  
Hormone System

BackgroundExposure to early life adversity (ELA) has been identified as a major risk factor in the development of major depressive disorder (MDD). It is hypothesized that a mediating mechanism may be environmentally induced alterations in gene function. In our REDEEM (Research in depression: endocrinology, epigenetics and neuroimaging) project we are examining possible epigenetic difference in some previously investigated target genes relevant to depression. To this end, methylation of the following genes were measured: NR3C1 (HPA axis), SLC6A4 (serotonin neurotransmitter function), and CD3ɛ (T cell receptor gene). We also looked at possible trans-generational transmission of epigenetic markers in a mother-baby sample.MethodsDNA was isolated from depressed patients and controls and babies and a portion of the above genes, encompassing our regions of interest, were amplified by PCR. Percentage methylation levels were measured by pyrosequencing. mRNA was also measured for some gene products to see if function was related to methylation. HPA axis function was measured with serial saliva samples throughout the day.Resultsto date: Methylation was increased in the CD3ɛ promoter in depressed subjects relative to controls. In the total group, those exposed to ELA had significantly increased methylation at this site. Levels of CD3ɛ mRNA levels were inversely related to methylation. There were some relationships between maternal ELA and baby methylation at the sites examined.ConclusionsConsistent with an allostatic model of ELA damage, our findings suggest an alteration in epigenetic function in acquired immunity and the HPA axis, mediated by ELA. Findings will be discussed.Disclosure of interestThe authors have not supplied their declaration of competing interest.

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