scholarly journals Differential Regulation of Oocyte Maturation and Cumulus Expansion in the Mouse Oocyte– Cumulus Cell Complex by Site-Selective Analogs of Cyclic Adenosine Monophosphate

1995 ◽  
Vol 172 (1) ◽  
pp. 72-85 ◽  
Author(s):  
Stephen M. Downs ◽  
Mary Hunzicker-Dunn
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Cell Complex ◽  
Mouse Oocyte ◽  
Cumulus Expansion ◽  
Site Selective

scholarly journals Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 535
Author(s):  
Nikoleta Pavlaki ◽  
Kirstie A. De Jong ◽  
Birgit Geertz ◽  
Viacheslav O. Nikolaev ◽  
Alexander Froese
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Membrane Microdomains ◽  
Transgenic Mouse Line

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

259 PRELIMINARY INVESTIGATIONS OF THE EFFECT OF NITRIC OXIDE ON THE EXPRESSION OF GENES INVOLVED IN THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE/CYCLIC ADENOSINE MONOPHOSPHATE PATHWAY DURING MEIOSIS RESUMPTION IN BOVINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 277
Author(s):  
K. R. L. Schwarz ◽  
M. R. Chiaratti ◽  
L. G. Mesquita ◽  
C. L. V. Leal
Keyword(s):  
Nitric Oxide ◽  
Oocyte Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Temporary Increase ◽  
Expression Of Genes ◽  
Genes Encoding

Nitric oxide is a chemical messenger generated by the activity of the enzyme NO synthase (NOS) and has been implicated in the control of oocyte maturation. Nitric oxide stimulates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cyclic adenosine monophosphate (cAMP) levels, a nucleotide also involved in meiosis resumption. In a previous study, we found that increasing NO levels in the presence of a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) resulted in a delayed resumption of meiosis and a lower rate of germinal vesicle breakdown after 9 h of in vitro maturation. A temporary increase in cGMP levels was also observed with the same treatment, which was reversed by inhibiting GC activity with oxadiazolo-quinoxaline-one (ODQ; unpublished data). These results suggest that NO acted via GC/cGMP and that even a temporary increase in the cGMP level led to a delay of meiosis resumption. The aim of the present study was to determine the role played by NO on the expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9 h of oocyte maturation. Cumulus–oocyte complexes were in vitro matured for 9 h in a semi-defined medium (TCM-199 + 3 mg mL–1 of BSA) with 10 to 7 M SNAP associated or not associated with 100 µM ODQ, a GC inhibitor. A group of oocytes incubated in the absence of inhibitors was considered the control. Total RNA was extracted from pools of 20 denuded oocytes with TRIzol (Life Technologies, Grand Island, NY, USA) and reverse transcribed into complementary DNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed by real-time PCR using SYBR Green (Applied Biosystems). The genes that had their expression measured pertained to one of the following groups: 1) genes encoding for enzymes that synthesise NO (NOS2 and NOS3); 2) genes involved in the control of cGMP levels (GUCY1B3 and PDE5A) or the enzymes activated by it (PKG1 and PKG2); or 3) genes involved in the control of cAMP levels (ADCY3, ADCY6, ADCY9, PDE3A, and PDE8A) or one of the enzymes activated by it (PKA1). GAPDH and PPIA were selected as housekeeping genes using qbasePLUS version 2.3 (Biogazelle, Zwijnaarde, Belgium). Data from 5 replicates were analysed using LinRegPCR version 11.1 and SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). All genes were found to be expressed in the three experimental groups; however, a significant difference in gene expression levels was not found among groups. Results suggest that NO does not act on oocyte maturation by affecting the expression of the investigated genes in oocytes. To our knowledge, this is the first report to demonstrate the expression of the ADCY3, ADCY6, and ADCY9 genes in bovine oocytes. Further research is in progress to study the effect of the SNAP treatment on the expression of these genes in cumulus cells. Financial support from FAPESP 2010/18023-9.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

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.

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