159 Effect of bisphenol A and bisphenol S on AMH and AMHR mRNA expression during in vitro bovine oocyte maturation and early embryo development

2019 ◽  
Vol 31 (1) ◽  
pp. 204 ◽  
Author(s):  
A. Saleh ◽  
L. Favetta
Keyword(s):  
Bisphenol A ◽  
Mrna Expression ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Bisphenol S ◽  
Tissue Samples ◽  
Receptor Mrna

Exposure to chemicals with known endocrine-disrupting effects, such as bisphenol A (BPA) and bisphenol S (BPS), leads to repercussions on oocyte development and, ultimately, on fertility. Bisphenol A is a plasticizer used worldwide that has been detected in blood, urine, tissue samples and follicular fluid. Due to its widely reported detrimental effects, BPA has been substituted with its analogue BPS. Previous experiments in our laboratory have shown that exposure of bovine oocytes to physiologically relevant doses of BPA resulted in spindle abnormalities, reduced meiosis progression, decreased blastocyst rate and gene expression changes. However, the effects of BPS have not yet been investigated. Anti-Müllerian hormone (AMH) has been reported to be a good marker of ovarian reserve and oocyte developmental capability and is commonly used in assisted reproduction for diagnostic measurements. There is evidence that women undergoing IVF with higher BPA levels have lower AMH levels and pregnancy success. The aim of this study was to assess the effect of BPA and BPS on AMH and its receptor as measures of oocyte developmental capability. Abattoir-derived bovine cumulus-oocyte complexes (COC) were matured in vitro in 4 groups: (1) control, (2) vehicle (0.1% ethanol), (3) BPA (0.05mg mL−1 in 0.1% ethanol), and (4) BPS (0.05mg mL−1 in 0.1% ethanol). Pools of 30 COC, 30 denuded oocytes, and cumulus cells corresponding to denuded oocytes were collected for each of the 4 experimental groups, and a minimum of 4 biological replicates were used for each analysis. Anti-Müllerian hormone and AMH receptor mRNA expression was measured in COC, denuded oocytes, and their corresponding cumulus cells using quantitative real-time PCR. Statistical analyses were performed using 1-way ANOVA. Results showed a decrease (P<0.05) in AMH mRNA expression in BPA-treated oocytes (without cumulus cells). In addition, there was an increase (P<0.05) in mRNA AMH receptor levels in COC when treated with BPS. Finally, analyses on cumulus cells alone showed an increase (P<0.05) in the AMH receptor mRNA levels in BPA-treated cells. These results suggest that BPA has an effect on AMH mRNA transcript levels in oocytes, while affecting the receptor expression in cumulus cells. Conversely, BPS affects AMH indirectly, increasing the mRNA levels of its receptor only. Further investigation of the effects of BPA and BPS on AMH expression in in vitro-produced blastocysts derived from treated oocytes will aid in understanding the potential consequences of exposure to BPA and its analogues on early embryonic development.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

2013 ◽  
Vol 25 (6) ◽  
pp. 890 ◽  
Author(s):  
Ester Siqueira Caixeta ◽  
Mariana Fernandes Machado ◽  
Paula Ripamonte ◽  
Christopher Price ◽  
José Buratini
Keyword(s):  
Mrna Expression ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Direct Stimulation ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Epidermal Growth

FSH induces expansion of bovine cumulus–oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.

249 EXPRESSION OF mRNA ENCODING EPIDERMAL GROWTH FACTOR-LIKE FACTORS IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FOLLICLE-STIMULATING HORMONE

2011 ◽  
Vol 23 (1) ◽  
pp. 222
Author(s):  
E. S. Caixeta ◽  
M. F. Machado ◽  
P. Ripamonte ◽  
P. F. Lima ◽  
A. C. S. Castilho ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Significant Difference ◽  
Epidermal Growth

Epidermal growth factor (EGF)-like family members [amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC)] have been shown to be important regulators of cumulus–oocyte complex (COC) maturation, particularly cumulus expansion. The aim of this study was to determine the temporal expression patterns of mRNA encoding EGF-like growth factors in bovine cumulus cells (CC) during COC in vitro maturation and to assess the effects of grading doses of FSH on EGF-like mRNA expression in CC. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries. In the first experiment, CC were separated from 20 COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 h with (10 ng mL–1) or without FSH. In the second experiment, pools containing 20 COC were matured for 12 h with grading doses of FSH (0, 0.1, 1, 10, and 100 ng mL–1). After culture, CC were mechanically separated and stored at –80°C. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed. Expression of target genes was assessed by real-time PCR and normalized by Cyclophilin (CYC-A). Relative quantification of mRNA abundance was determined by the Pfaffl equation. Effects of time of culture and FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer honestly significant difference test. Nonparametric analysis was used when data were not normally distributed. Differences were considered significant when P < 0.05. In the presence of FSH, AREG and EREG mRNA abundance was increased at 4 h of culture, whereas in the absence of FSH, AREG but not EREG mRNA levels were increased by 4 h of culture. The addition of FSH stimulated AREG mRNA expression from 4 to 16 h of culture. In contrast, BTC mRNA was more expressed in immature CC, decreased after 4 h of culture with FSH, and did not vary during maturation in the absence of FSH. In the dose–response experiment, AREG and EREG mRNA expression was stimulated by FSH starting from 10 ng mL–1 and did not increase from 10 ng mL–1 to 100 ng mL–1. Again in contrast, BTC mRNA expression was inhibited by FSH at 100 ng mL–1. In conclusion, the present data suggest that FSH differently regulates the expression of EGF-like factors during bovine COC maturation, although AREG and EREG are stimulated, BTC is inhibited by FSH. This work was supported by FAPESP.

scholarly journals A GH receptor antisense oligonucleotide inhibits hepatic GH receptor expression, IGF-I production and body weight gain in normal mice

2006 ◽  
Vol 189 (1) ◽  
pp. 147-154 ◽  
Author(s):  
G Tachas ◽  
S Lofthouse ◽  
C J Wraight ◽  
B F Baker ◽  
N B Sioufi ◽  
...  
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Antisense Oligonucleotide ◽  
Body Weight Gain ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Receptor Mrna ◽  
Gh Receptor ◽  
Igf I

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2′-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

Bone morphogenetic protein-6 (BMP-6): mRNA expression and effect on steroidogenesis during in vitro maturation of porcine cumulus oocyte complexes

2011 ◽  
Vol 23 (8) ◽  
pp. 1034 ◽  
Author(s):  
S. Ebeling ◽  
D. Töpfer ◽  
J. M. Weitzel ◽  
B. Meinecke
Keyword(s):  
Mrna Expression ◽  
In Vitro Maturation ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Levels ◽  
17Β Estradiol ◽  
Estradiol Synthesis

Oocyte secreted factors (OSFs) have emerged as important factors for follicular development. The present study investigated the effect of the potential OSF bone morphogenic protein (BMP)-6 on steroidogenesis in porcine cumulus oocyte complexes during in vitro maturation. Cumulus oocyte complexes (COCs), cumulus complexes (CCs) without oocytes and CCs with supplemented BMP-6 were cultured for 0, 5, 26 or 46 h. BMP-6 transcripts were detected in oocytes and cumulus cells at all time points. In both cell types the mRNA expression was most intense after 5 h, and decreased during further maturation. After 26 and 46 h of culture, CCs secreted significantly less 17β-estradiol than COCs. This effect was reversed by adding BMP-6 to CCs cultures. In addition, a down-regulation of Cyp19A1, the rate-limiting enzyme of 17β-estradiol synthesis, was detected in CC cultures after 5 h. As seen for 17β-estradiol secretion, the addition of BMP-6 caused a significant increase in Cyp19A1 mRNA levels after 5, 26 and 46 h of culture. Progesterone secretion and transcripts of steroidogenic marker proteins StAR and 3β-HSD were not affected considerably by oocyte removal or addition of BMP-6. Furthermore, BMP-6 did not affect the activity of the mitogen-activated protein kinase. The results indicated that BMP-6 is a potential OSF and is involved in the prevention of premature luteinisation in cumulus cells via enhancing 17β-estradiol synthesis.

scholarly journals Regulation of nerve growth factor receptor gene expression in sympathetic neurons during development.

1995 ◽  
Vol 130 (6) ◽  
pp. 1435-1446 ◽  
Author(s):  
S Wyatt ◽  
A M Davies
Keyword(s):  
Mrna Expression ◽  
Sensory Neurons ◽  
Sympathetic Neurons ◽  
Receptor Gene ◽  
Receptor Expression ◽  
Early Developmental Stage ◽  
Mrna Levels ◽  
Fos Expression

We used quantitative reverse transcription (RT)/PCR to study the regulation of p75 mRNA and trkA mRNA expression in the developing sympathetic neurons of the mouse superior cervical sympathetic ganglion (SCG) in vivo and in vitro. At E13, the SCG contains proliferating cells that express many features of differentiated neurons. These immature neurons survived in culture without NGF, and NGF did not induce c-fos expression. Low levels of p75 and trkA mRNAs were expressed at this stage in vivo. There was no significant increase in the level of either trkA mRNA or p75 mRNA in E13 control cultures up to 72 h in vitro, and neither NGF nor depolarizing levels of K+ ions (40 mM KC1) affected the expression of trkA mRNA. In E14 cultures, NGF induced c-fos expression in 10-15% of the neurons and enhanced the survival of a similar percentage of neurons. The proportion of neurons responding to NGF increased with age, reaching 90% in E18 cultures. The in vivo level of trkA mRNA increased markedly from E14 onward, but in contrast to sensory neurons (in which p75 and trkA mRNA levels increase in parallel), the level of trkA mRNA initially increased far more rapidly than that of p75 mRNA. After E17, the level of p75 mRNA increased rapidly and approached that of trkA mRNA postnatally, but at no stage did this exceed the level of trkA mRNA. In E14 cultures, the level of trkA mRNA increased in the absence of neurotrophins or 40 mM KC1. The level of p75 mRNA in E14 cultures was enhanced by NGF but was unaffected by 40 mM KC1. Our findings show that NGF receptor expression during the earliest stages of sympathetic neuron development is not affected by depolarization but indicate that by an early developmental stage (between E13 and E14 in vivo), sympathetic neurons become specified to upregulate trkA mRNA in culture independently of added factors. In addition, our findings reveal several distinctive features of p75 mRNA and trkA mRNA expression in sympathetic neurons compared with sensory neurons and provide a plausible explanation for previously observed differences in the effects of a p75 null mutation on the response of sensory and sympathetic neurons during embryonic and postnatal development.

Effect of Estrogen on Melanocortin-3 Receptor mRNA Expression in Mouse Pituitary Glands in vivo and in vitro

2004 ◽  
Vol 80 (3) ◽  
pp. 143-151 ◽  
Author(s):  
Ryusei Matsumura ◽  
Sakae Takeuchi ◽  
Sumio Takahashi
Keyword(s):  
Mrna Expression ◽  
Receptor Mrna ◽  
Pituitary Glands ◽  
Mouse Pituitary

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

scholarly journals Regulation of interleukin 6 receptor expression in human monocytes and monocyte-derived macrophages. Comparison with the expression in human hepatocytes.

1989 ◽  
Vol 170 (5) ◽  
pp. 1537-1549 ◽  
Author(s):  
J Bauer ◽  
T M Bauer ◽  
T Kalb ◽  
T Taga ◽  
G Lengyel ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Human Peripheral Blood ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Blood Monocytes ◽  
Inflammatory Conditions ◽  
High Concentrations ◽  
Human Primary Hepatocytes ◽  
Stimulation Of

IL-6 is a cytokine with pleiotropic biological functions, including induction of the hepatic acute phase response and differentiation of activated B cells into Ig-secreting plasma cells. We found that human peripheral blood monocytes express the IL-6-R, which is undetectable on the large majority of lymphocytes of healthy individuals. Stimulation of monocytes by endotoxin or IL-1 causes a rapid downregulation of IL-6-R mRNA levels and a concomitant enhancement of IL-6 mRNA expression. IL-6 itself was found to suppress the IL-6-R at high concentrations. A gradual decrease of IL-6-R mRNA levels was observed along in vitro maturation of monocytes into macrophages. We show that downregulation of IL-6-R mRNA levels by IL-1 and IL-6 is monocyte specific, since IL-6-R expression is stimulated by both IL-1 and IL-6 in cultured human primary hepatocytes. Our data indicate that under noninflammatory conditions, monocytes may play a role in binding of trace amounts of circulating IL-6. Repression of monocytic IL-6-R and stimulation of hepatocytic IL-6-R synthesis may represent a shift of the IL-6 tissue targets under inflammatory conditions.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice

2012 ◽  
Vol 303 (10) ◽  
pp. L852-L860 ◽  
Author(s):  
S. Yoshida ◽  
N. Minematsu ◽  
S. Chubachi ◽  
H. Nakamura ◽  
M. Miyazaki ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Apoptosis ◽  
Pulmonary Emphysema ◽  
Keratinocyte Growth Factor ◽  
Annexin V ◽  
Phenotypic Change ◽  
Chronic Obstructive ◽  
Mrna Levels

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Activation of nuclear factor of activated T cells 5 in the peritoneal membrane of uremic patients

2015 ◽  
Vol 308 (11) ◽  
pp. F1247-F1258 ◽  
Author(s):  
Daniel Kitterer ◽  
Joerg Latus ◽  
Christoph Ulmer ◽  
Peter Fritz ◽  
Dagmar Biegger ◽  
...  
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Significant Difference ◽  
Ccl2 Mrna ◽  
Uremic Patients ◽  
High Concentrations

Peritoneal inflammation and fibrosis are responses to the uremic milieu and exposure to hyperosmolar dialysis fluids in patients on peritoneal dialysis. Cells respond to high osmolarity via the transcription factor nuclear factor of activated T cells (NFAT5). In the present study, the response of human peritoneal fibroblasts to glucose was analyzed in vitro. Expression levels of NFAT5 and chemokine (C-C motif) ligand (CCL2) mRNA were quantified in peritoneal biopsies of five nonuremic control patients, five uremic patients before PD (pPD), and eight patients on PD (oPD) using real-time PCR. Biopsies from 5 control patients, 25 pPD patients, and 25 oPD patients were investigated using immunohistochemistry to detect the expression of NFAT5, CCL2, NF-κB p50, NF-κB p65, and CD68. High glucose concentrations led to an early, dose-dependent induction of NFAT5 mRNA in human peritoneal fibroblasts. CCL2 mRNA expression was upregulated by high concentrations of glucose after 6 h, but, most notably, a concentration-dependent induction of CCL2 was present after 96 h. In human peritoneal biopsies, NFAT5 mRNA levels were increased in uremic patients compared with nonuremic control patients. No significant difference was found between the pPD group and oPD group. CCL2 mRNA expression was higher in the oPD group. Immunohistochemistry analysis was consistent with the results of mRNA analysis. CD68-positive cells were significantly increased in the oPD group. In conclusion, uremia results in NFAT5 induction, which might promote early changes of the peritoneum. Upregulation of NFAT5 in PD patients is associated with NFκB induction, potentially resulting in the recruitment of macrophages.

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