268 CYCLIC GUANOSINE MONOPHOSPHATE INHIBITS PHOSPHODIESTERASE 3 ACTIVITY IN PORCINE OOCYTES

Abstract

The level of cyclic adenosine monophosphate (cAMP) within oocytes has been shown to play a critical role in maintaining meiotic arrest. High levels of intracellular cAMP prevent spontaneous oocyte maturation in vitro, whereas a decrease in oocyte cAMP is associated with the resumption of meiosis. Another cyclic nucleotide that also was recently proposed as being involved in meiotic resumption is cyclic guanosine monophosphate (cGMP), which could be regulating phosphodiesterase (PDE) 3 activity. The aim of this study was to determine whether cGMP inhibits cAMP-PDE activity in porcine oocytes. With the method described previously by Sasseville et al. (2006 BMC Dev. Biol. 6, 47), PDE activity was measured in groups of 10 oocytes cultured in the absence (control) or presence of different concentrations of cGMP (1, 3, 10, 30, 100, 300, 1000, and 3000 nM) or with the PDE3 inhibitor cilostamide (10 µM). Before assaying PDE activity, the cumulus–oocyte complexes (COC) were matured in vitro for 24 h in the presence of pregnant mare serum gonadotropin (5 IU) and hCG (5 IU) at 38.5°C in 5% CO2. The COC were homogenised in a hypotonic buffer. Data were analysed using one-way ANOVA followed by Duncan’s post-hoc test. Differences with P < 0.05 were considered significant. Results showed that 300, 1000, and 3000 nM cGMP inhibited PDE3 activity (7.9, 5.1, and 4.1 fmol min–1 per COC; P < 0.05) at levels below the controls (13.2 fmol min–1 per COC) and were similar to the activity observed in the presence of (2.4 fmol min–1 per COC; P > 0.05). The other concentrations tested were similar to activity levels seen in the control (1 to 100 nM; 12.2, 11.3, 10.8, 11.5, and 10.4; P > 0.05). In conclusion, the results support the concept that increasing concentrations of cGMP inhibit PDE activity, suggesting the inhibition of the predominant form of cAMP-PDE present in porcine oocytes, PDE3. These results support the hypothesis that cGMP inhibits PDE activity in porcine oocytes. Further work is needed to determine the role this plays in maintaining high cAMP levels and inhibiting oocyte nuclear maturation. Financial support from FGZ FAPESP 2010/20188-6 and 2010/18023-9 is acknowledged.