PARENT SESSION
Oocyte Development
83
BOVINE OOCYTE MEIOTIC MATURATION AND GRANULOSA CELL cAMP LEVELS ARE DIFFERENTIALLY REGULATED BY TYPE-SPECIFIC PHOSPHODIESTERASE INHIBITORS.
Gilchrist, Robert1, Thomas, Rebecca1, Armstrong, David1, 1
ABSTRACT- Bovine oocyte meiotic maturation is regulated in part by changing ooplasmic and cumulus cell cAMP levels which may be differentially regulated by cell-type specific phosphodiesterases (PDE). This study was conducted to examine the effect of type-specific PDE inhibitors on bovine oocyte maturation and granulosa cell function. Ovaries were obtained from an abattoir and cumulus-oocyte complexes (COC) and mural granulosa cells (MGC) were aspirated from antral follicles into culture medium supplemented with 4 mg/ml BSA and 2 mM 3-isobutyl-1-methylxanthine (IBMX). COC or denuded oocytes (DO) were cultured either with and without forskolin (FK) in the presence of selective PDE inhibitors; either milrinone (MR, PDE3 inhibitor), rolipram (RP, PDE4 inhibitor) or IBMX (non-specific PDE inhibitor). MGC were subjected to the same treatments as oocytes except with the addition of FSH. Cyclic AMP production from 24h MGC cultures was assessed by RIA. COC and DO were fixed after 16h of culture and assessed for meiotic progression. In the presence of FSH, MGC cAMP levels were significantly (P<0.001) elevated by RP and IBMX (2.5 and 4.0 fold respectively) above control levels. However, cAMP levels were unaffected by MR, demonstrating that cAMP in bovine MGC is regulated by type 4 and not type 3 PDE. In contrast, oocyte meiotic maturation was affected by the type 3, but not by the type 4 PDE inhibitor. In the absence of FK, MR but not RP slightly inhibited meiotic resumption (germinal vesicle breakdown, GVB) compared to controls (P<0.05; 87, 99 and 100% GVB, respectively). Although FK alone only marginally reduced GVB (90%), the action of FK was significantly enhanced in the presence of MR and RP (57 and 62% GVB, respectively). Both FK and MR by themselves markedly inhibited the completion of meiosis to metaphase II (MII) compared to controls (25, 44 and 84% MII, respectively), and the inhibitory effect of FK was further enhanced by MR (9%) but not by RP (34%). Similar results were obtained with DO except RP + FK was less inhibitory in DO (91% GVB, 54% MII) than in COC. Together these results demonstrate that there is selective localisation of PDE subtypes between the germ cell and somatic cell compartments within bovine follicles, and suggest that oocyte maturation is inhibited more effectively by the oocyte than the somatic cell-specific PDE inhibitor.
KEY WORDS: oocyte maturation, phosphodiesterase inhibitor, granulosa cell, cAMP, oocyte meiosis