176. INHIBITION OF PORCINE OOCYTE NUCLEAR MATURATION IN VITRO USING A PHOSPHODIESTERASE INHIBITOR AND AN ADENYLATE CYCLASE ACTIVATOR

2010 ◽  
Vol 22 (9) ◽  
pp. 94
Author(s):  
M. Bertoldo ◽  
T. Sellens ◽  
C. G. Grupen
Keyword(s):  
Adenylate Cyclase ◽  
Phosphodiesterase Inhibitor ◽  
Phosphodiesterase Inhibitors ◽  
Extended Period ◽  
Nuclear Maturation ◽  
Porcine Oocyte ◽  
Defined Culture ◽  
Cyclase Activator ◽  
Adenylate Cyclase Activator

Asynchronous nuclear and cytoplasmic maturation is thought to contribute to poor embryo production in vitro. Nuclear arrest is mediated by cAMP and can be maintained within the oocyte using non-specific phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine ; IBMX) and the adenylate cyclase activator forskolin (FSK) (1). The aim of this study was to investigate the effect of IBMX and FSK supplementation on porcine oocyte nuclear maturation during COC recovery and IVM using a defined culture system. In all experiments, cAMP modulators were added to Hepes-buffered media held in collection tubes. COCs recovered from 3–5 mm diameter follicles of prepubertal ovaries were cultured in basic maturation media in the absence of FSH. Nuclear maturation was assessed using orcein dye. In Experiment 1, IVM media was supplemented with 0, 50 or 500 µM IBMX. In Experiment 2, IVM media was supplemented with 0, 5, 10, 50 and 100µM FSK. In Experiment 3, IVM medium was supplemented with combinations of IBMX and FSK to give the treatments; control, 50IBMX/50FSK, 50IBMX/100FSK, 500IBMX/50FSK and 500IBMX/100FSK. Nuclear maturation was assessed at 0, 2, 4 and 18 h after the onset of IVM. At 18 h of culture, there were no differences in the proportion of oocytes supplemented with 0, 50 or 500 µM IBMX reaching MII. Incubation with 10, 50 or 100 µM FSK resulted in 8-16% of oocytes at MII at 18 h compared to the other groups (25–29%; P < 0.001). The combinations of IBMX and FSK resulted in greater proportions (86–98%) of oocytes remaining at the GV stage at 18 h compared to the control (16%; P < 0.001). There were no differences in the proportion of oocytes remaining at the GV stage at the earlier time points (P > 0.05). The results demonstrate that these cAMP modulators, in combination, are highly effective in maintaining porcine oocyte meiotic arrest in vitro for an extended period. (1) Albuz FK et al., Proceedings of the 25th Annual Meeting of ESHRE, Amsterdam, The Netherlands, 2009.

Conditions to optimise the developmental competence of immature equine oocytes

2020 ◽  
Vol 32 (11) ◽  
pp. 1012
Author(s):  
Elizabeth S. Metcalf ◽  
Keith R. Masterson ◽  
David Battaglia ◽  
Jeremy G. Thompson ◽  
Robert Foss ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Culture Media ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Sodium Ascorbate ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Holding Temperature ◽  
Cyclase Activator ◽  
Adenylate Cyclase Activator

Optimising the developmental potential of immature equine oocytes and invitro-produced (IVP) embryos was explored through modifications of established media and holding temperature. In Experiment 1, delaying spontaneous resumption of meiosis through the process of simulated physiological oocyte maturation with the addition of the adenylate cyclase activator forskolin (50µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100µM) to overnight holding medium before maturation improved blastocyst production (P&lt;0.05). In Experiment 2, the blastocyst production rate was increased significantly when cumulin (100ng mL−1) was added to the overnight holding or culture media (P&lt;0.05). In Experiment 3, immature oocytes held overnight at 16°C before maturation had improved developmental competence than those held at 20°C and 5°C (P&lt;0.05). There was no difference between maturation rates, but blastocyst formation per cleaved oocyte was significantly greater in oocytes held overnight at 16°C than at 20°C or 5°C. Furthermore, blastocyst formation per recovered oocyte and per fertilised oocyte was greater when oocytes were held before maturation at 16°C than at 5°C (P&lt;0.05). In Experiment 4, the addition of sodium ascorbate (AC; 50µg mL−1) to the maturation and/or culture media of oocytes and IVP embryos did not improve blastocyst production, but did appear to lower cleavage rates compared with oocytes and embryos cultured without AC.

356 MODULATION OF PORCINE NUCLEAR MATURATION STATUS USING A SPECIFIC PHOSPHODIESTERASE INHIBITOR

2010 ◽  
Vol 22 (1) ◽  
pp. 334
Author(s):  
M. L. Sutton-McDowall ◽  
R. B. Gilchrist ◽  
J. G. Thompson
Keyword(s):  
Gap Junction ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Adenosine Monophosphate ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Gap Junction Communication ◽  
Defined Culture ◽  
Type 3

Compared to other livestock species, IVM porcine COCs have poor developmental competence.Thisismost likely due to poor cytoplasmic maturation and asynchronous nuclear maturation. The resumption of nuclear maturation is largely regulated by cyclic adenosine monophosphate(cAMP), with increasing intra-oocyte levels prolonging cumulus-oocyte gap junction communication and delaying meiotic resumption. Modulation of cAMP levels using phosphodiesterase (PDE) inhibitors during bovine IVM significantly improves developmental competence (Thomas et al. 2004 Biol. Reprod. 71, 1142-1149). Hence, the aim of this study was to determine the effect of a type 3 PDE inhibitor (cilostamide, CIL) supplementation during porcine IVM on nuclear maturation, using a defined culture system. COCs derived from follicles of prepubertal gilts were cultured in groups of 10 in 100μL IVM medium (VitroMat, IVF Vet Solutions, Adelaide, Australia) +100 m IU mL-1 rhFSH +4 mg mL-1 BSA and nuclear maturation was assessed using orcein dye. Exp. 1: IVM medium was supplemented with 0, 5, 10, and 20 μM CIL or 0, 0.01, 0.1, 1, and 10 μM CIL and nuclear maturation was determined at 24 h and 44 h. Exp. 2: COCs were cultured in ± 0.1 μM CIL and nuclear maturation was assessed at 24 h, 44 h, 48 h, 52 h, and 56 h. Proportions of COCs at each stage of nuclear maturation were arcsine transformed and differences determined using a general linear model and Bonferroni post hoc test. Four replicates per experiment were performed with 20 COCs used for each treatment and/or time point. Results for Exp. 1 revealed no differences in the rate of nuclear maturation after 24 h of culture. After 44 h, 77% of COCs incubated in the absence of CIL were at metaphase II (MII) compared to 35-45% MII when cultured in the presence of 1, 5, 10, or 20 μM CIL (P < 0.001). Furthermore, CIL supplementation resulted in approximately half the COCs arresting at germinal vesicle stage (GV) after 44 h of culture. While there were no significant differences in the MII rates of COCs cultured in 0, 0.01, and 0.1 μM CIL, significantly more COCs were at metaphase I (MI) at 44 h compared to control COCs (0 μM CIL, P < 0.05). The time course experiment (Exp 2) demonstrated that nuclear maturation was delayed by 12 h with 0.1 μM CIL, compared to the absence of CIL, with comparable MII rates being achieved at 56 h for 0.1 μM CIL (72%) and 44 h for controls (0 μM CIL = 73%). These results demonstrate that porcine oocyte maturation can be induced in vitro by FSH in the presence of a low dose of type 3 PDE inhibitor resulting in a delay in the resumption and completion of nuclear maturation. Further investigations are underway to determine if CIL treatment prolongs gap junction communication and improves the developmental competence of porcine oocytes. This work was supported by the National Institutes of Health (USA) and National Health and Medical Research Council (Australia).

Circadian change in function of Limulus ventral photoreceptors

1988 ◽  
Vol 1 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Leonard Kass ◽  
George H. Renninger
Keyword(s):  
Circadian Clock ◽  
Phosphodiesterase Inhibitor ◽  
Pharmacological Agents ◽  
Lateral Eye ◽  
Intracellular Microelectrodes ◽  
Circadian Change ◽  
Cyclase Activator ◽  
Adenylate Cyclase Activator

AbstractEfferent fibers from a central circadian clock innervate photoreceptors along the ventral nerve of Limulus and release octopamine when active. We have recorded ERG-like responses from the ventral eye in vivo over several day periods. We have also used intracellular microelectrodes to study changes in ventral photoreceptor function during exogenous applications of octopamine (the putative efferent neurotransmitter), IBMX (a phosphodiesterase inhibitor), and forskolin (an adenylate cyclase activator): (1) Responses to light measured at night from ventral photoreceptors in vivo are greater in amplitude than those recorded during the day; (2) Octopamine and agents that increase intracellular levels of cAMP in ventral photoreceptors decrease the rate of spontaneous (dark) bumps, increase photoreceptor response to light without changing threshold, and often increase the bump duration; and (3) These changes in function of ventral photoreceptors are similar to those that have been observed in the photoreceptor of the lateral eye during circadian clock activity at night, and in vitro in the presence of those same pharmacological agents.

Increase in protein and tubulin mRNA synthesis in frog sensory neurons treated with the adenylate cyclase activator, forskolin

1990 ◽  
Vol 1 (3,4) ◽  
pp. 225-232
Author(s):  
Richard C. Carlsen ◽  
Marino De Leon ◽  
Wolfram Tetzlaff ◽  
Irma M. Parhad ◽  
Mark A. Bisby
Keyword(s):  
Adenylate Cyclase ◽  
Sensory Neurons ◽  
Mrna Synthesis ◽  
Tubulin Mrna ◽  
Cyclase Activator ◽  
Adenylate Cyclase Activator

Reversible effects of phenothiazines on frog gastric stimulation

1984 ◽  
Vol 247 (4) ◽  
pp. G366-G376
Author(s):  
N. Raphael ◽  
E. B. Ekblad ◽  
T. E. Machen
Keyword(s):  
Adenylate Cyclase ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Secretory Activity ◽  
Gastric Stimulation ◽  
Camp Content ◽  
Oxyntic Cell ◽  
Camp Generation ◽  
Stimulus Secretion Coupling

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and promethazine (PZ) were tested for effects on stimulus-secretion coupling in in vitro bullfrog gastric mucosa. When added to histamine-stimulated tissues, the drugs caused H+ secretion to decrease and transepithelial resistance to increase over a 2-h time course. The potency sequence was TFP (IC50 = 40 microM) greater than CPZ (IC50 = 72 microM) congruent to PZ (IC50 = 72 microM). Anesthetics and other phenothiazines with weak anticalmodulin activity had no effect on secretory parameters. In the presence of histamine, further addition of isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) plus dibutyryl cAMP (DBcAMP), IBMX alone, or forskolin (a specific activator of adenylate cyclase) to phenothiazine-inhibited tissues caused full resumption of secretory activity. If TFP (50 microM) was added before stimulation with histamine, the normal increases in tissue cAMP content (which occurs primarily in oxyntic cells), oxyntic cell apical membrane elaboration (morphometric analysis of electron micrographs), and H+ secretion were all blocked. Subsequent addition of IBMX or IBMX plus DBcAMP completely reversed the TFP effect. These results indicate that the histamine-sensitive adenylate cyclase may be the site of TFP inhibition and Ca2+-calmodulin regulation; since these drugs inhibited stimulation by DBcAMP plus IBMX, they may also be exerting additional effects distal to cAMP generation.

Effect of lanosterol on the in vitro maturation in semi-defined culture system of prepubertal ewe oocytes

Zygote ◽  
2011 ◽  
Vol 22 (1) ◽  
pp. 50-57 ◽  
Author(s):  
F. Marco-Jiménez ◽  
J.S. Vicente ◽  
M.P. Viudes-de-Castro
Keyword(s):  
Oxygen Concentration ◽  
Cell Function ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Low Oxygen ◽  
Oocyte Growth ◽  
Important Indicator ◽  
Nuclear Maturation ◽  
Defined Culture

SummaryThe choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 106 and 7.4 ± 0.2 × 106 arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 106 arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.

208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 235
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Treatment Group ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Formation Rate ◽  
Transforming Growth Factor Β ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

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