Co-incubation of oocytes with the selective phosphodiesterase 9 (PDE9) inhibitor BAY 73-6691 (BAY) and low levels of cGMP blocks spontaneous resumption of meiosis in vitro

Abstract

OBJECTIVE: A drop in cAMP triggered by PDE 3 is a key step in meiotic resumption of the oocyte. cGMP is an endogenous inhibitor of PDE 3, and is known to prevent oocyte maturation at high levels. The objective for this study was to determine if a PDE9 (cGMP-specific phosphodiesterase expressed in the oocyte) inhibitor could inhibit spontaneous resumption of meiosis in oocytes. DESIGN: Controlled rodent dose response experiments. MATERIALS AND METHODS: Germinal vesicle (GV) intact oocytes collected from PMG-stimulated BDF-1 mice and stripped of cumulus cells, were incubated in KSOM media containing either BAY (10 μM, 100 μM, or 1 mM), 8-Br-cGMP, [the membrane-permeable analogue of cGMP, (500 μM, 1 mM, or 10 mM)], a combination of both treatments [8-Br-cGMP (1 mM) and either 100 μM or 1 mM BAY], or no additional treatment (control). Oocytes were evaluated for GV-breakdown (GVBD) as an indicator of meiotic resumption after 20-22 hours. Results were analyzed using two-tailed Fisher's exact test. RESULTS: In total, 46/49 (94%) control oocytes progressed to GVBD, compared with 40/48 (83%, P = 0.76), 20/60 (33%, P < 0.001) and 0/54 (0%, P < 0.001) in oocytes incubated with 8-Br-cGMP at 500 μM, 1 mM, or 10 mM respectively. While culture of oocytes in any concentration of BAY alone did not significantly affect meiosis, co-culture with either 1mM or 100 μM BAY and 1 mM 8-Br-cGMP prevented GVBD in the majority of oocytes (92 & 93% respectively; p<0.001). Interestingly, none of the oocytes which resumed meiosis in co-culture matured to metaphase II, but rather arrested at metaphase I, whereas 23% of the oocytes cultured in only 1mM 8-Br-cGMP progressed to MII. CONCLUSION: While a PDE9 inhibitor alone is not sufficient to inhibit oocyte maturation in vitro, a powerful effect is seen in combination with added cGMP. Therefore a combined approach that increases cGMP transport to the oocyte from granulosa cells and blocks degradation with a PDE9 inhibitor may have contraceptive potential.