Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle1

Abstract

The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5' nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.

Regulation of sheep oocyte maturation using cAMP modulators

“…Comparatively, SPOM pre-IVM in bovine generated a 100-fold increase in cAMP concentrations [13]. Apparent differences between the two species may be due to the presence and expression of different PDE and adenylate cyclase isoforms [11]. However, since cAMP concentrations do not substantially differ between ovine and bovine COCs in the presence of IBMX and FSK (< 20 fmol/COC), perhaps peripubertal ovine COCs have greater endogenous production of cAMP, or ovine PDEs degrade cAMP at a slower rate than in bovine COCs.…”

“…This was expected due to differences among species in PDE kinetics, the presence of other PDE subtypes within the oocyte, and varying concentrations of endogenous oocyte cAMP production [46]. In the pre-IVM phase, 10-fold higher concentrations of IBMX were needed in bovine compared to murine models (500 versus 50 μM, respectively), perhaps due to expression of PDE8 in bovine COCs, which accounted for more than 20% of total PDE activity and is not inhibited by IBMX [11]. Conversely, murine cumulus cells did not express PDE8, but expressed the IBMXsensitive PDE4 as its major cumulus cell PDE [12].…”

“…However, there are major differences among species with regards to cAMP degrading phosphodiesterase (PDE) isoforms [11,12]. Maintenance of cAMP concentrations within an oocyte is achieved through a balance of production (synthesis of cAMP with adenylate cyclases and GPR), degradation (mainly the result of PDEs), and cAMP transfer from CCs.…”

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte

“…In that system, treatment with the uncoupler 1-heptanol induced abrupt chromatin condensation and decreased in transcription. However, the addition of the oocyte-specific PDE3 inhibitor cilostamide [31][32][33] to the culture medium nullified this effect indicating that the functional status of GJC may affect both transcriptional activity and remodeling of largescale chromatin configuration in growing oocytes, potentially through cAMP-dependent mechanism(s) [19].…”

Reversible meiotic arrest in feline oocytes

“…Both of these chemicals have been used extensively in multiple species with consistent, predicted effects (Bilodeau et al 1993;Fan et al 2002;Thomas et al 2002;Chen et al 2009;Downs 2011). In mice, rats, pigs, cattle, macaques and humans, the majority of PDE activity within the oocyte is due to PDE3, which is sensitive to IBMX (Downs et al 1989;Conti et al 1998;Richard et al 2001;Jensen et al 2002;Thomas et al 2002;Liang et al 2005;Nogueira et al 2006;Sasseville et al 2009). By inhibiting PDE3 activity, IBMX maintains meiotic arrest by preventing a decline in cAMP concentrations within the oocyte after recovery (Schultz et al 1983;Luciano et al 2004;Albuz et al 2010) or permitting the accumulation of cAMP produced by intra-oocyte adenylate cyclase(s) (Mehlmann 2005).…”