Identification of phosphodiesterase 9A as a cyclic guanosine monophosphate-specific phosphodiesterase in germinal vesicle oocytes: a proposed role in the resumption of meiosis

Abstract

Objective: To identify a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE) in nonhuman primate germinal vesicle (GV) oocytes and establish a proposed effect on oocyte maturation through preliminary experiments in mouse GV oocytes.

Design: Controlled nonhuman primate and rodent experiments.

Setting: Academic research institution.

Animal(s): Rhesus macaques and B6/129F1 mice.

Intervention(s): Stimulation of Rhesus macaques with follicle-stimulating hormone (FSH) to collect GV oocytes and cumulus for gene expression analysis, and stimulation of female mice with pregnant mare serum gonadotropin to collect GV oocytes.

Main outcome measure(s): Expression of PDE transcript in primate GV oocytes and cumulus cells, measurement of fluorescence polarization of phosphodiesterase 3A (PDE3A) activity, and analysis of spontaneous resumption of meiosis in mouse GV oocytes.

Result(s): Of five PDE transcripts detected in Rhesus GV oocytes, only PDE9A was cGMP-specific. The fluorescence polarization assays indicated cGMP has an inhibitory effect on PDE3A while the phosphodiesterase 9A (PDE9) inhibitor, BAY73-6691, does not. Similarly, BAY73-6691 had little effect on preventing spontaneous maturation in oocytes, but did augment the inhibitory effects of cGMP. Inclusion of 0 μM (control), 10 μM, 100 μM, and 1 mM BAY73-6691 statistically significantly increased the proportion of mouse oocytes maintaining GV arrest in the presence of the cGMP analog 8-Br-cGMP at 100 μM (8.8%, 11.4%, 18.8%, and 28%), 500 μM (21.1%, 38.1%, 74.5%, and 66.5%), and 1 mM (57.8%, 74.5%, 93.9%, and 94.0%), respectively.

Conclusion(s): Phosphodiesterase 9A (PDE9A) is a cGMP-specific hydrolyzing enzyme present in primate oocytes, and PDE9 antagonists augment the inhibitory effect of cGMP during spontaneous in vitro maturation of GV mouse oocytes.

Figure 1

Quantitative PCR (qPCR) profiling of PDE expression in rhesus monkey GV oocytes and granulosa cells collected from pre-ovulatory antral follicles. (A) Of the 19 PDE genes that were assayed, only PDE1C, PDE3A, PDE8A, PDE8B, and PDE9A were detectable in the GV oocyte. (B) PDE expression in granulosa cells, PDE11A was the only isoform not detectable in any of the samples evaluated. Expression ratios are based on a comparison to the endogenous control gene; 18s-rRNA for oocytes and RPS10 for granulosa cells. Error bars indicate standard deviation. One Way ANOVA was performed with a Student-Newman-Keuls post hoc test to determine significant changes in expression with P < 0.001. * Indicates expression is significantly less than all other isoforms. ** Expression is significantly greater than all other isoforms.

Figure 2

Florescence polarization assay for PDE3A activity. Increasing concentrations of the PDE9 inhibitor, BAY 73-6691, were evaluated for inhibitory properties against PDE3A activity at 0 µM (no inhibitor), 1 µM, 10 µM, 100 µM, and 1 mM (Red). Similarly, the cGMP analog, 8-Br-cGMP, was assayed at 0 µM (no inhibitor), 1 µM, 10 µM, 100 µM, 1 mM and 10 mM (Blue). No significant changes were observed for the PDE9 inhibitor. Different letters indicate a significant decrease in PDE3A and Z’ = 0.75 for both assays. Significant changes in activity were analyzed by ANOVA followed by posthoc comparison with Student-Newman-Keuls test with P<0.001. Error bars indicate the first standard deviation.

Figure 3

Percentage of mouse oocytes that retained the GV after 20–22 hours of culture with (A) the selective PDE9 inhibitor BAY 73-6691, or (B) a combination of both 8-Br-cGMP and BAY 73-6691. Error bars represent the standard deviation; value in parenthesis represents the total number of oocytes in each treatment group for (A). Different letters at each level of 8-Br-cGMP in (B) represent a significant change from the control and determine by Chi Square analysis with P<0.001.

Figure 4

Proposed PDE9 activity pathway in the oocyte. (A) cGMP from granulosa cells is actively transported through gap junctions into the GV oocyte. Elevated levels of cytoplasmic cGMP inhibit PDE3A hydrolysis of cAMP and maintain the oocyte in meiotic arrest. Based on our data, following the LH surge, gap junctions are closed, and residual cGMP is degraded by PDE9 (B). Decreased levels of cGMP permit PDE3A mediated hydrolysis of cAMP, lowering the cytoplasmic concentration and inducing meiotic resumption.