Cilostamide, a phosphodiesterase 3A inhibitor, sustains meiotic arrest of rat oocytes by modulating cyclic adenosine monophosphate level and the key regulators of maturation promoting factor.

Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.

Meiotic arrest as an alternative to increase the production of bovine embryos by somatic cell nuclear transfer.

This study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal-Wallis test with a P-value 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

The effect of pre-maturation culture using phosphodiesterase type 3 inhibitor and insulin, transferrin and selenium on nuclear and cytoplasmic maturation of bovine oocytes.

This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 µM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 µM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 µM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

The glucose lowering effects of CL 316,243 dissipate with repeated use and are rescued bycilostamide.

Repeated activation of the beta 3 adrenergic receptor (ß3AR) with the agonist CL 316,243 (CL) results in remodeling of white adipose tissue (WAT) characterized by increased mitochondrial enzymes and expression of uncoupling protein 1 (UCP1). ß3AR activation also has profound acute metabolic effects including rapidly decreasing blood glucose, secondary to fatty acid-induced increases in insulin, and increasing energy expenditure. The acute (single dose) effects of ß3AR activation have largely been examined in treatment naive animals and under room temperature housing conditions. The current study examined if repeated CL treatment would lead to an attenuation of acute metabolic effects of CL treatment under thermoneutral housing conditions and if this could be rescued with cilostamide, a phosphodiesterase inhibitor. We provide evidence demonstrating that the acute effects of CL to increase serum fatty acids and insulin and reduce blood glucose, but not increases in energy expenditure, are attenuated in mice following repeated treatment with CL. This occurs in parallel with reductions in indices of protein kinase A signaling in WAT including the phosphorylation of hormone sensitive lipase. The findings of attenuated serum fatty acid, insulin, and blood glucose responses were confirmed in both high-fat fed and UCP1-/- mice repeatedly treated with CL. Desensitization to CL in mice was rescued by cilostamide. Herein, we provide evidence that the glucose lowering, but not thermogenesis inducing, effects of CL are attenuated with repeated treatment and can be rescued by cilostamide. The findings of this study point toward novel adjunct treatment approaches that could be used to maximize therapeutic, glucose lowering effects of ß3AR agonists.

Cilostamide and forskolin maintain gap junction function of incubated dog follicles.

Disruption of the communication between the oocyte and granulosa cells is one of the major causes of poor development of in vitro grown ovarian follicles and oocytes. The present study investigated the effect of two cAMP modulators, cilostamide and forskolin, on in vitro growth of isolated dog secondary follicles and enclosed oocytes, communication between the gamete and surrounding granulosa cells, expression of GJA1 and GDF9, as well as cAMP level. Secondary follicles were incubated with cilostamide or forskolin alone or a combination of 20 µM cilostamide +1 µM forskolin, and the diameter of the incubated follicles and enclosed oocytes assessed every 72 h. Gap junction activity, GJA1 and GDF9 expression and cAMP level were assessed on Days 6 and 12 and transzonal projection (TZP) density was evaluated on Day 12. Neither cilostamide nor forskolin alone enhanced in vitro growth of dog follicles and the enclosed oocytes (P > 0.05). However, these two cAMP modulators dose dependently sustained gap junction activity and stimulated cAMP production compared with the non-supplemented control. Cilostamide at the high dosage (20 µM) also upregulated GJA1 expression. The combination of cilostamide and forskolin supported oocyte growth during the first 9 days and upregulated GJA1 and GDF9 expression at Day 12 of in vitro culture. This combination treatment also sustained gap junction activity, cAMP production, and increased TZP function (calcein intensity: TZP density ratio). The findings indicated that a combination of cilostamide and forskolin supported growth and survival of oocytes enclosed within cultured follicles by sustaining cAMP production and gap junction activity.

The SPOM-adapted IVM system improves in vitro production of bovine embryos.

This study aimed to test the effects of an IVM SPOM adaptation (SPOM-adapted IVM) on the production, total number of cells (TNC), apoptosis, and cryotolerance (post-warming survival and cytoskeleton actin integrity) of bovine IVP embryos. Two experiments were conducted with two experimental groups based on IVM treatment: A control group (TCM 199 without FCS) and an SPOM-adapted group (TCM 199 with forskolin and IBMX in pre-IVM and IVM with cilostamide). The first experiment evaluated embryo in vitro production, TNC, and apoptosis rate on D9 of development. In the second experiment, embryos were vitrified/warmed at D7 (control fresh and vitrified; SPOM-adapted fresh and vitrified) and assessed regarding post-warming survival rates and cytoskeleton actin integrity. Statistical analysis was performed using GraphPad INSTAT software at a significance level of 5%. An increase (p < 0.05) in blastocyst production was observed in the SPOM-adapted group comparing to the control group. There was no difference (p > 0.05) in the TNC or apoptosis rate between the groups. Regarding cryopreservation, no differences were found (p > 0.05) in actin integrity or post-warming survival rates between the vitrified groups. In both vitrified groups, we observed a significantly lower uninjured pattern of actin integrity compared to the fresh groups (p < 0.05). We conclude that the SPOM-adapted IVM system is beneficial for blastocyst production and does not affect the quality and cryotolerance of the produced embryos.

Cilostamide and rolipram prevent spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro.

The involvement of specific phosphodiesterases (PDEs) in the modulation of cAMP and thereby spontaneous meiotic resumption remains poorly understood. This work aims to evaluate the effects of cilostamide and rolipram (PDE 3A and PDE 4D inhibitors) on spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro. For this purpose, diplotene-arrested cumulus oocyte complexes (COCs) were collected from rat ovary. The COCs and denuded oocytes were exposed to various concentrations of cilostamide (0.0, 2.5, 5.0 and 10 µM) and rolipram (0, 10, 50 and 100 µM) for various times (0, 3, 5, 7, 14, 16, 18, 20, 22 and 24 h). Cilostamide inhibited spontaneous meiotic resumption in a concentration- and time-dependent manner in COCs and denuded oocytes. Although rolipram showed inhibition of spontaneous meiotic resumption up to some extent, cilostamide was more potent to prevent spontaneous meiotic resumption in both COCs and denuded oocytes. Cilostamide significantly reduced PDE 3A expression, increased cAMP level and prevented spontaneous meiotic resumption in COCs and denuded oocytes. Although rolipram inhibited PDE 4D expression in cumulus cells, increased cAMP level but was not sufficient to prevent spontaneous meiotic resumption. We conclude that both drugs prevent spontaneous resumption from diplotene-arrest through PDE 3A/PDE 4D-cAMP mediated pathway. However, as compare to rolipram, cilostamide was more potent in preventing spontaneous resumption from diplotene-arrest in rat oocytes cultured in vitro. Thus, cilostamide could be used as a potential candidate for the development of female contraceptive drug in future.

Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes.

This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 µM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 µM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.

Endolymphatic hydrops induced by different mechanisms responds differentially to spironolactone: a rationale for understanding the diversity of treatment responses in hydropic inner ear disease.

Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice. Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways. Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry. Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium. Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others. Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.

Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI.

CONCLUSION: The data indicate important roles for phosphodiesterase (PDE) 3, 4, 5, and related cAMP and cGMP pools in the regulation of inner ear fluid homeostasis. Thus, dysfunction of these enzymes might contribute to pathologies of the inner ear. OBJECTIVE: The mechanisms underlying endolymphatic hydrops, a hallmark of inner ear dysfunction, are not known in detail; however, altered balance in cAMP and cGMP signaling systems appears to be involved. Key components of these systems are PDEs, enzymes that modulate the amplitude, duration, termination, and specificity of cAMP and cGMP signaling. METHOD: To evaluate the role of PDE3, 4, and 5 and associated cAMP and cGMP pools in inner ear function, the effect of cilostamide (PDE3 inhibitor), rolipram (PDE4 inhibitor), and sildenafil (PDE5 inhibitor), administrated via mini-osmotic pumps, on mouse inner ear fluid homeostasis was evaluated using 9.4T in vivo MRI in combination with intraperitoneally administered Gadolinium contrast. Also, using human saccule as a model, the expression of PDEs and related signaling molecules and targets was studied using immunohistochemistry. RESULTS: PDE3, PDE4, as well as PDE5 inhibitors resulted in the development of endolymphatic hydrops. Furthermore, PDE3B, PDE4D, and some related signaling components were shown to be expressed in the human saccule.

Supplement of cilostamide in growth medium improves oocyte maturation and developmental competence of embryos derived from small antral follicles in pigs.

This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles < 3 mm in diameter (SAFCOCs). SAFCOCs were untreated (control) or treated with 20 µM CIL and/or 50 µM FSK for 2 days for IVG. Next, IVG oocytes were cultured for maturation and then induced for parthenogenesis (PA) or used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation of oocytes was significantly lower in the control (49.6 ± 9.3%) group than in other groups (67.2 ± 5.0-79.8 ± 7.9%). The cumulus expansion score after IVG-IVM was significantly higher in the control and CIL group than in the FSK and CIL + FSK groups. CIL significantly increased mean diameter SAF-derived oocytes (120.0 ± 0.5 µm) compared to the control, FSK, and CIL + FSK (114.8 ± 0.5-116.7 ± 0.6 µm) and showed a comparable level of intracellular glutathione (GSH) contents (0.84 ± 0.07 pixels/oocyte) to medium antral follicle (MAF)-derived oocytes (1.00 ± 0.08 pixels/oocyte), but was higher than those of oocytes treated with FSK and CIL + FSK (0.29 ± 0.05 and 0.37 ± 0.05 pixels/oocyte, respectively). CIL treatment significantly increased blastocyst formation (55.1 ± 4.7%) after PA relative to the control (29.4 ± 6.4%), FSK (34.8 ± 7.1%), and CIL + FSK (41.1 ± 5.2%). A higher proportion of oocytes treated with CIL, FSK, and CIL + FSK (73.3 ± 1.7-82.8 ± 9.1%) remained at the germinal vesicle stage after IVG culture than control oocytes (40.0 ± 5.0%). Following SCNT, blastocyst formation of SAFCOCs treated with CIL (22.4 ± 6.3%) was higher than that of oocytes (0-10.4 ± 5.3%) in control, FSK, and CIL + FSK, but similar to that (25.3 ± 3.5%) of MAF-derived COCs not cultured for IVG. The cAMP level of SAFCOCs before IVG was 0.1 ± 0.03 fmol/oocyte. After 2 days of IVG culture, cAMP level was increased significantly by treatment with FSK and CIL + FSK (3.0 ± 0.57 and 12.1 ± 0.62 fmol/oocyte, respectively) relative to the control and CIL treatment (0.1 ± 0.03 and 0.3 ± 0.04 fmol/oocyte, respectively). Our results demonstrate that CIL treatment during IVG improves the low developmental competence of SAFCOCs to levels comparable to MAFCOCs by allowing oocyte growth while inhibiting premature meiotic maturation, probably via maintenance of cAMP concentrations at appropriate levels.

Novel cilostamide analogs, phosphodiesterase 3 inhibitors, produce positive inotropic but differential lusitropic and chronotropic effects on isolated rat atria.

OBJECTIVES: Recently, we showed that some new synthetic compounds structurally related to cilostamide (4-(1,2-dihydro-2-oxoquinolin-6-hydroxy)- N-cyclohexyl-N-methylbutanamide), a selective phosphodiesterase 3 (PDE3) inhibitor, produce inotropic effect comparable to that of IBMX (3-isobutyl-1-methylxanthine), a non-selective PDE inhibitor, but with differential chronotropic effect. In this investigation, we compared the pharmacological effects of these compounds as potential cardiotonic agents using the spontaneously beating atria model. MATERIALS AND METHODS: In each experiment, rats were treated with reserpine. The atrium was isolated and mounted in an organ bath. We assessed chronotropic and inotropic effects using cumulative log concentration-response curves of isoprenaline alone or in combination of each test-compound. RESULTS: Majority of test compounds augment atria contraction force (ACF) significantly but with different potencies on atrium contraction rate. Cilostamide, MCPIP ([4-(4-methyl piperazin-1-yl)-4-oxobutoxy)-4-methylquinolin-2(1H)-one]), methyl carbostyril compounds- (mc1), mc2 and mc5 increased the isoprenaline effect on ACF synergistically. But, mc6 failed to potentiate the effect of isoprenalin; mc3 and mc4 did not increase ACF, which may be because of their higher hydrophilic nature. It was interesting that mc2, alone or in combination with isoprenaline, produced the highest inotropic effect while it did not affect the basal contraction rate and almost blocked the isoprenaline chronotropic effect. CONCLUSION: Combination of mc2 with isoprenaline had synergistic effect on inotropic effect, but this combination reduced isoprenaline chronotropic effect; therefore, these effects cannot be related to reducing B-adrenergic receptors activity. These compounds showed different effects; probably all of them were not mediated via PDE3 inhibition and other mechanisms are involving.

Effect of prematuration and maturation with fibroblast growth factor 10 (FGF10) on in vitro development of bovine oocytes.

In an attempt to improve in vitro embryo production, we investigated the effect of FGF10 and 0.1 mM of cilostamide, cAMP modulator, during in vitro prematuration (PIVM) and in vitro maturation (IVM) on the developmental capacity of bovine oocytes. Five treatments (T) were used as follows: T1) IVM (control): COCs were matured for 22 h; T2) PIVM/IVM: COCs were submitted to 22 h of PIVM and 22 h of IVM; T3) PIVM + FGF10/IVM: COCs were submitted to PIVM for 22 h in the presence of FGF10 and matured for 22 h; T4) PIVM/IVM + FGF10: COCs were submitted to PIVM for 22 h and matured for 22 h in the presence of FGF10; and T5) PIVM + FGF10/IVM + FGF10: COCs were submitted to 22 h of PIVM and 22 h of IVM, both in the presence of FGF10. COCs were evaluated for CC expansion and nucleus configuration at 0 h, 22 h of PIVM and 22 h after IVM. At those time points, CCs were used for expression analysis of PTGS2, TNFAIP6, PTX3, HAS2, CASP8, CASP3, SLC2A1, SLC2A3 and FGFR2 genes. Then, embryo production was evaluated on D2 for cleavage and at D6, D7 and D8 for embryo development. Embryo quality was measured by the speed of development and by expression of KRT8, PLAC8, CD9, PAG2, PAG2, HSPB1, MSH6 genes. The percentage of COC that remained at GV after 22 h PIVM was lower (P < 0.05) in the treated group than in the control group, regardless of the addition of FGF10. Nevertheless at 22 h of maturation, none of the treatments affected the final rate of MII oocytes. No expansion of CCs was observed during the PIVM period. After 22 h of maturation expansion was similar (P > 0.05) for all groups but they all had lower expansion (P < 0.05) than control group. Except for PTGS2 and SLC2A1 which were unchanged during PIVM, all other genes changed their expression during PIVM. When we evaluated the changes in gene expression during IVM on the different groups, we noted a change in the expression of four genes (PTGS2, CASP8, PTX3 and TNFAIP6). No differences (P > 0.05) in the cleavage and blastocyst rates at D6 were observed among treatment groups. However, the blastocyst rates at D7 were lower (P < 0.05) than the control groups in which FGF10 was present either during PIVM or during IVM. When the speed of embryo development was evaluated on D7, embryos from groups PIVM-IVM and PIVM - IVM + FGF10 developed faster than the other groups. However, at D8, the hatching rate was similar (P > 0.05) for all groups. Of all the analysed genes, only PLAC8 was shown to be overexpressed in PIVM/IVM + FGF10, and MSH6 was less expressed (P < 0.05) in PIVM + FGF10/IVM + FGF10 group when compared with the control. According to these findings, the PIVM of COCs in the presence of FGF10 affected the molecular profile and the expansion of CCs without affecting the nuclear maturation and embryo production rates. Although the presence of FGF10 changed the expression of genes related to embryo quality it did not show a great impact when added either or both during PIVM and IVM.

cAMP modulation during sheep in vitro oocyte maturation delays progression of meiosis without affecting oocyte parthenogenetic developmental competence.

Removal of oocytes from their natural inhibitory follicular environment results in spontaneous resumption of meiosis independent of normal signaling events that occur in vivo. Controlling the onset of meiotic resumption via maintenance of elevated oocyte cAMP levels with adenylyl cyclase (AC) activation and phosphodiesterase (PDE) inhibition, and subsequent hormone stimulation with follicle FSH has been shown to dramatically improve developmental competence of bovine and murine IVM oocytes. This study evaluated the effect of cAMP modulation during IVM of sheep oocytes on meiotic progression and development to blastocyst after parthenogenetic activation. Changes in oocyte cAMP levels were quantified during the first 2h of in vitro maturation in control or cAMP-modulating medium. No significant changes in intra-oocyte cAMP were observed under control conditions, though a slight and transient drop was noticed at 15 min of maturation. Addition of the AC stimulator Forskolin and the PDE inhibitors IBMX altered the cAMP profile, resulting in 10-fold elevation of cAMP by 15 min and sustained >3-fold elevated levels from 30 to 120 min. The effect of cAMP elevation on meiotic resumption was measured by completion of germinal vesicle breakdown. Modulated oocytes were significantly delayed when compared to control media oocytes. Also, progression to MII was significantly delayed in modulated versus control oocytes at 20 and 24h, though no differences persisted to 28 h. Lastly, when control and modulated oocytes were parthenogenetically activated, no differences in blastocyst formation were observed. Thus, while cAMP modulation delayed meiotic progression, it did not improve developmental competence of sheep IVM oocytes.

Does levosimendan act as a Ca2+ sensitizer or PDE3 inhibitor?: Commentary on Orstavik et al., Br J Pharmacol 171: 5169-5181.

LINKED ARTICLE: This article is a Commentary on Orstavik O, Ata SH, Riise J, Dahl CP, Andersen GO, Levy FO, Skomedal T, Osnes J-B, and Qvigstad E (2014). PDE3-inhibition by levosimendan is sufficient to account for its inotropic effect in failing human heart . Br J Pharmacol 171: 5169-5181. doi: 10.1111/bph.12647.

Differential metabolic effects of novel cilostamide analogs, methyl carbostiryl derivatives, on mouse and hyperglycemic rat.

OBJECTIVE(S): PDE3 has a functional role in insulin secretion and action. We investigated the metabolic effects of new synthetic PDE3 inhibitors (mc1, mc2, mc5 and mc6), on mice and hyperglycemic rat. MATERIALS AND METHODS: The test compound or solvent was injected subcutaneously to mice, for 7 days. On day 8, blood and liver samples were obtained. In hyperglycemic rat, 0.5 g/kg glucose with or without test compounds was injected, and followed with infusion of 1.5 g/kg/hr glucose. Blood samples were collected in mentioned intervals and liver was dissected. RESULTS: In hyperglycemic rat, all test compounds decreased blood glucose and the effect of milrinone was potentiated by glybenclamide. Milrinone or IBMX did not change plasma insulin levels, but it was augmented by combination of milrinone and glybenclamide. In both species, liver glycogen storage was decreased by IBMX, mc5, mc6 or MCPIP, increased by mc2 (liver glycogen, rat, control=56±2, mc2=70±3 P< 0.01, mice, control=33±0.7, mc2=42±2.3 P< 0.01) and was not changed in the presence of mc1. Milrinone did not change the glycogen storage in rats though increased it in mice (control= 33±0.7, milrinone= 40±1 P< 0.05). CONCLUSION: Increasing plasma insulin levels by combination of milrinone and glybenclamide confirmed that in hyperglycemic rat, the hypoglycemic effect was correlated with increasing insulin secretion. Variations of plasma insulin were obscured by the pulsative characteristic of pancreatic insulin release. Decreasing glycogen storage reflected inhibition of liver PDE activity. The reasons for ineffectiveness of mc1, anabolic effect of mc2, and differential effects of milrinone were not clear.