scholarly journals 285 REGULATION OF OOCYTE MEIOTIC RESUMPTION USING cAMP MODULATORS IN BOVINE IN VITRO MATURATION

2015 ◽  
Vol 27 (1) ◽  
pp. 231 ◽  
Author(s):  
S. E. Farmer ◽  
J. A. Sarmiento-Guzmán ◽  
C. L. Bailey ◽  
K. R. Bondioli
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Current Experiment ◽  
Negative Effect ◽  
Low Efficiency ◽  
Camp Levels

In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP). Currently, IVP has low efficiency due to an inadequate IVM system where premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3′,5′-cyclic adenosine monophosphate (cAMP). Some successful methods of improving IVM have utilised cAMP modulators to maintain high intraoocyte cAMP, delaying the onset of nuclear maturation and allowing cytoplasmic maturation to occur. The current experiment is a follow-up to previous work in which an extended 2-step maturation system was examined. In the previous experiments, we found that meiotic resumption was significantly delayed, but the overall maturation rates of extended IVM were about half those of standard IVM, suggesting that the effects of the modulators on the oocytes were not completely reversible. The current experiment compares cAMP concentrations of oocytes in this extended IVM to standard IVM to determine whether high cAMP is the cause of the low maturation rate. Bovine oocytes (n = 686) were obtained from mixed-breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into 2 groups: standard IVM and extended IVM. Standard IVM consists of a 23-h maturation composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured at 39°C in 5% CO2. Extended IVM consists of 2 steps: a pre-IVM phase composed of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 h at 39°C, followed by an extended IVM phase composed of standard IVM media supplemented with 20 µM cilostamide for 31 h (39°C, 5% CO2). Additionally, oocytes in the extended IVM treatment group where held in HEPES-TALP media with FSK and IBMX during the 2-h oocyte collection period in order to prevent any decrease in cAMP before the oocytes could be placed in the extended IVM media. Oocytes in standard IVM were sampled at 0, 8, and 23 h of maturation, while oocytes in extended IVM were sampled at 0, 8, 18, and 33 h of maturation. Cumulus cells were removed from all oocytes by vortexing in hyaluronidase solution. Oocytes were stored in groups of 10 at –80°C. A cAMP enzyme immunoassay (GE Healthcare) was performed to determine cAMP concentrations throughout standard and extended IVM. Assay results were analysed using an ANOVA followed by a Tukey's pairwise test (Sigma Stat 3.5) to detect significant differences (P < 0.05). Results indicated significantly higher cAMP levels in extended IVM oocytes during the first 3 h after collection using FSK and IBMX in the holding media (0.505 v. 1.006 fmol/oocyte, P = 0.035) but cAMP levels were not maintained in the cilostamide-only extended IVM medium. This suggests that high cAMP levels were not the cause of low maturation rates in extended IVM, since cAMP concentrations did decrease after 3 h. Possible negative effect of cilostamide on these oocytes may be suggested and need to be analysed.

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

Treatment with cyclic adenosine monophosphate modulators prior to in vitro maturation alters the lipid composition and transcript profile of bovine cumulus–oocyte complexes and blastocysts

2018 ◽  
Vol 30 (10) ◽  
pp. 1314 ◽  
Author(s):  
Eduardo M. Razza ◽  
Mateus J. Sudano ◽  
Patricia K. Fontes ◽  
Fernanda F. Franchi ◽  
Katia Roberta A. Belaz ◽  
...  
Keyword(s):  
Lipid Content ◽  
Lipid Composition ◽  
Transcriptional Profiling ◽  
Ovarian Follicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Membrane Lipid ◽  
Unsaturated Lipids

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus–oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2 h and 16 transcripts after 24 h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

258 IMPACT OF MILRINONE AND FORSKOLIN ON THE EFFICIENCY AND QUALITY OF BOVINE OOCYTE IN VITRO MATURATION

2013 ◽  
Vol 25 (1) ◽  
pp. 277
Author(s):  
E. Stachowiak ◽  
K. Papis ◽  
J. Karasiewicz ◽  
J. A. Modlinski
Keyword(s):  
In Vitro Maturation ◽  
Combined Treatment ◽  
Phosphodiesterase Inhibitor ◽  
Microscopic Analysis ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Experimental Conditions ◽  
Oocyte Meiosis

The efficiency of in vitro maturation (IVM) of bovine oocytes remains inferior compared with maturation in vivo. Recently, some modifications of in vitro maturation (IVM) procedures have been proposed, such as simulated physiological maturation (Gilchrist 2011 Reprod. Fertil. Dev. 23, 23–31). In our experiment, a comparison of the traditional IVM efficiency with maturation after oocyte meiosis inhibition using roscovitine or with a modified two-step maturation using forskolin (cyclic adenosine monophosphate stimulator) and milrinone (type-3 phosphodiesterase inhibitor) was performed. Control oocytes obtained from slaughterhouse-derived ovaries were subjected to the traditional 24-h maturation in TCM-199 medium supplemented with sodium pyruvate, l-glutamine, gentamicin, 10% FCS, and hormones (pregnant mare’s serum gonadotropin and hCG, PG 600, Intervet, Kenilworth, NJ, USA). The roscovitine (50 µM, 24 h) inhibitory treatment was accomplished in the same medium (without hormones) and subsequently, traditional 24-h IVM was performed. The same TCM-199 medium (with hormones) supplemented with forskolin (100 µM) and milrinone (50 µM) was used for the first step (17 h) of the two-step maturation, whereas the second step (7 h) was performed in the same TCM-199 medium devoid of forskolin and milrinone. Fertilization with frozen sperm processed using TALP media was performed in TALP supplemented with heparin, penicillamine, hypotaurine, epinephrine, and BSA. In vitro culture of presumptive zygotes was performed in CR1aa medium. Portions of oocytes from all treatments after maturation and after fertilization procedures were stained and subjected to microscopic analysis. There were no differences in terms of maturation and fertilization rates between treatments. However, roscovitine-mediated inhibition of maturation performed in our experimental conditions was efficient and reversible, but harmful for subsequent embryo development. On the other hand, two-step maturation was equally as efficient as (but not better than) traditional IVM in all aspects examined in the present study (Table 1). In conclusion, the forskolin and milrinone combined treatment during the IVM procedure gives hope for fully efficient IVM. However, to achieve this goal, more research is necessary. Table 1.Development of embryos after different oocyte maturation procedures1

Effect of dibutyryl cyclic adenosine monophosphate on reactive oxygen species and glutathione of porcine oocytes, apoptosis of cumulus cells, and embryonic development

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 305-313 ◽  
Author(s):  
Sang-Hyoun Park ◽  
Il-Jeoung Yu
Keyword(s):  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Reactive Oxygen ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Oxygen Species ◽  
Cell Numbers

SummaryThe present study was conducted to investigate the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) supplemented into porcine maturation medium on reactive oxygen species (ROS) and glutathione (GSH) levels of oocytes, and apoptosis of cumulus cells (CC). In addition, the effect of dbcAMP on embryonic development following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Cumulus–oocyte complexes (COCs) were cultured in 0 mM (control), 0.5 mM, 1 mM, 5 mM, or 10 mM dbcAMP-supplemented medium for 22 h, then for another 22 h without dbcAMP. GSH and ROS levels of oocytes were assessed at 44 h of culture by dichlorohydrofluorescein diacetate or 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin staining, respectively. Additionally, COCs were cultured in 0.5 mM or 1 mM dbcAMP and then fertilized in vitro or activated parthenogenetically. Embryonic development and blastocyst cell numbers and apoptosis levels on day 8 of culture were investigated. CC apoptosis at 44 h of culture and blastocyst apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. GSH levels in the 0.5 mM dbcAMP and control groups were increased (P < 0.05), while levels of oocyte ROS and CC apoptosis in the control, 0.5 mM, and 1 mM dbcAMP groups were significantly lower than the levels in other groups. Cleavage and blastocyst rates, cell numbers, and apoptosis levels were not significantly different in embryos derived by either IVF or PA among the groups, with the exception of significantly increased apoptotic levels in IVF blastocysts produced from oocytes treated with 1 mM dbcAMP. In conclusion, dbcAMP treatment during in vitro maturation (IVM) did not improve embryonic development under our study's parameters compared with control conditions, although 0.5 mM dbcAMP showed significantly higher GSH levels and lower blastocyst apoptotic levels compared with 1 mM dbcAMP.

Genetic Polymorphisms of the ß2-Adrenergic Receptor Relate to Guanosine Protein-coupled Stimulator Receptor Dysfunction in Fibromyalgia Syndrome

2011 ◽  
Vol 38 (6) ◽  
pp. 1095-1103 ◽  
Author(s):  
YANGMING XIAO ◽  
WEIJING HE ◽  
I. JON RUSSELL
Keyword(s):  
Adrenergic Receptor ◽  
Fibromyalgia Syndrome ◽  
Mononuclear Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Intracellular Camp ◽  
Peripheral Blood Mononuclear ◽  
Genotype Frequencies ◽  
Camp Levels

Objective.To determine the genotype frequencies of ß2-adrenergic receptor (ß2AR) gene polymorphisms (Gly16Arg, Glu27Gln) in patients with fibromyalgia syndrome (FM) by comparison with unrelated healthy controls. We sought any clinical association with these polymorphisms and determined whether the polymorphisms would associate with a biologic guanosine protein-coupled stimulator receptor (Gs) dysfunction in FM.Methods.Study subjects included 97 clinically characterized patients with FM and 59 controls. The ß2AR polymorphisms at codons 16 and 27 were determined using polymerase chain reaction-restriction fragment length polymorphism. The Gs functions of peripheral blood mononuclear cells (PBMC) were tested using isoproterenol (ISO) as the adrenergic Gs ligand and measuring intracellular cyclic adenosine monophosphate (cAMP) levels.Results.The frequency of the ß2AR gene polymorphism Gly16Arg in FM (43.5%) was significantly lower than in controls (63.2%), suggesting that this genotype might have some effect on the risk of developing FM. The only clinical association in FM was with sleep dysfunction. Patients with FM who carried the ß2AR polymorphism Arg16Arg also exhibited significantly lower PBMC basal cAMP levels (p < 0.05) and lower ISO-stimulated cAMP levels (p < 0.05) than FM carrying Gly16Gly or Gly16Arg.Conclusion.This confirms a relationship between ß2AR polymorphism and FM. It is the first study to demonstrate ß2AR polymorphism-related differences in intracellular cAMP responses of FM PBMC after ß2AR stimulationin vitro. These findings may explain some of the differences in responsiveness of FM subgroups to the adrenergic agonist medications currently approved for FM treatment.

238 THE ROLE OF PHOSPHODIESTERASE (PDE) 5A AS MEDIATOR OF LIPOLYSIS IN BOVINE OOCYTES AND ITS EFFECT ON THE DEVELOPMENT AND LIPID CONTENT OF EMBRYOS PRODUCED IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 208
Author(s):  
K. R. L. Schwarz ◽  
P. R. Adona ◽  
R. C. Botigelli ◽  
M. Del Collado ◽  
C. Elias ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Content ◽  
Pde5 Inhibitor ◽  
Nile Red ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Significance Level ◽  
Bovine Oocytes

Intracellular levels of cyclic adenosine monophosphate modulators (cAMP) and cGMP, in adipocytes, are important for the regulation of the lipolysis rate. The phosphodiesterases (PDE) control cGMP and cAMP levels by degradation. Different PDE isoforms are expressed in bovine oocytes and cumulus cells. Previously, we found that using an inhibitor of PDE5A (sildenafil, SILD) increased cGMP levels in bovine oocytes during in vitro maturation (IVM). In the current study we investigated if inhibition of PDE5A during maturation reduces the lipid content in IVF embryos. For this, oocytes were cultured for 24 h in maturation medium with 10% FCS and 10–7 M SILD (treatment I), 10% FCS (treatment II) and 0.4% BSA (control; N ± 160 COC/groups submit to IVF). After COC were in vitro fertilized, cleavage (Day 4) and blastocyst rates (Day 7) were measured. Blastocysts were stained with Nile Red (1 μg mL–1) for lipid content quantification, by mean fluorescence intensity per μm2, measured in the ImageJ program (fluorescence intensity, f.i.). Four replicates were transformed to log10 and subjected to statistical analysis using the SAS system (SAS Institute Inc., Cary, NC, USA) by ANOVA followed by Tukey test with a significance level of 5%. No difference in cleavage (Day 4) and blastocyst (Day 7) rates were observed in all groups (82 and 41.9%, respectively), showing that presence of FCS, SILD, or both in IVM medium did not affect embryo development. Treatment I had higher lipid content (40.35 f.i.) than treatment II (31.12 f.i.), which in turn was also superior to control (22.31 f.i.). According to the results, the presence of FCS in IVM media generates embryos with higher lipid content, and association of FCS and SIL further increased lipid content. Although inhibition of PDE5 increases cGMP levels and leads to higher lipolysis, such an effect was not observed when SIL was used as the PDE5 inhibitor. Reasons for such findings are still unclear, but a possibility would be the activation of a negative feedback mechanism by the increased cGMP generated by SIL, because this nucleotide activates PKG, which in turn inhibits cGMP synthesis by guanylate cyclase. During development the lower cGMP levels could reduce lipolysis, resulting in increased lipid accumulation in embryos. Further studies are needed to address this possibility.

scholarly journals Comparative In Vitro Study of Various α2-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

2020 ◽  
Vol 9 (3) ◽  
pp. 809
Author(s):  
Guillaume Porta Bonete ◽  
Anne Godier ◽  
Pascale Gaussem ◽  
Tiphaine Belleville-Rolland ◽  
Alexandre Leuci ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Camp Pathway ◽  
P2y12 Receptor Antagonist ◽  
Induced Aggregation ◽  
Camp Levels ◽  
Platelet Functions

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y12 receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α2-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α2-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α2-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α2-agonists. Our results support the development of specific full and systemic α2-adrenoreceptor agonists for ticagrelor reversal.

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