Cyclic GMP signaling is involved in the luteinizing hormone-dependent meiotic maturation of mouse oocytes

Abstract

It is well established that cAMP signaling is an important regulator of the oocyte meiotic cell cycle. Conversely, the function of cGMP during oocyte maturation is less clear. Herein, we evaluated the expression of cGMP-hydrolyzing phosphodiesterases (PDEs) in the somatic and germ cell compartments of the mouse ovarian follicle and demonstrate that PDE5 is preferentially expressed in somatic cells. Cyclic GMP is a potent inhibitor of cAMP hydrolysis from oocyte extracts, with a 50% inhibitory concentration of 97 nM. Luteinizing hormone (LH) stimulation of cultured preovulatory follicles results in a marked decrease in cGMP content, and a nadir is reached in 1.5 h; similarly, oocyte cGMP levels decrease after gonadotropin stimulation in vivo. The LH-dependent decrease in cGMP requires activation of the epidermal growth factor network. Treatment of follicles with a PDE5 inhibitor increases cGMP in the follicle well above unstimulated levels. Although LH causes a decrease in cGMP in follicles preincubated with PDE5 inhibitors, the levels of this nucleotide remain above unstimulated levels. Under these conditions of elevated cGMP, LH stimulation does not cause oocyte maturation after 5 h of incubation. Microinjection of a cGMP-specific PDE into oocytes causes meiotic maturation of wild-type oocytes, suggesting that an intraoocyte pool of cGMP is involved in the maintenance of meiotic arrest. This effect is absent in PDE3A-deficient oocytes. Taken together, these findings provide evidence that cGMP and cAMP signaling cooperate in maintaining meiotic arrest via regulation of PDE3A and that a decrease in cGMP in the somatic compartment is one of the signals contributing to meiotic maturation.

Figures

FIG. 1.

Cyclic GMP inhibits PDE3 activity in DOs. Denuded oocytes were isolated from 24-day-old mice after 42–44 h of stimulation with 5 IU of eCG. The cAMP-PDE activity was measured in total oocyte extract as described in Materials and Methods. The equivalent of 20 oocytes was used per point. Activity was measured in the presence of increasing concentrations of cGMP (1.5–15 000 nM), and 4000 nM cilostamide (CILO) was used as a positive control for PDE3 inhibition. Data are the mean ± range of two independent experiments performed in triplicate. Activity is expressed as femtomoles of cAMP hydrolyzed per minute per oocyte.

FIG. 2.

Cyclic GMP PDE activity in whole ovary, follicles, and DOs. Ovaries, POFs, and DOs were collected from 24-day-old mice after 42–44 h of stimulation with 5 IU of eCG. The cGMP-PDE activity was measured on total cell extracts as described in Materials and Methods. Final concentrations of inhibitors are indicated. A and B) Activity in the ovary (A) and POFs (B). Activity is expressed in picomoles of cGMP hydrolyzed per minute per milligram of total protein for ovary and POFs. The plus and minus signs indicate the presence (+) and absence (−) of the indicated compounds. C) Cyclic GMP PDE activity in femtomoles of cGMP hydrolyzed per minute in relation to oocyte number. D) The effects of inhibitors on the oocyte cGMP PDE activity. Activity in D is expressed as a percentage, where total activity is 0.39 fmol of cGMP hydrolyzed per minute per oocyte. Data are the mean ± SEM of at least four independent experiments.

FIG. 3.

Time course of cGMP accumulation in the preovulatory follicle exposed to LH and effect of hCG on oocyte cGMP levels. A) Preovulatory follicles were dissected from eCG-primed immature mice. After 30 min of equilibration in vitro under the conditions detailed in Materials and Methods, rLH at a final concentration of 5 IU was added at Time 0. At the times indicated on the abscissa, follicles were harvested, frozen in liquid nitrogen, and stored until use. For the cGMP assay, extracts were processed as detailed in Materials and Methods, and the cGMP content was measured by ELISA with acetylated samples. Filled and open squares represent follicles with and without LH treatment, respectively. The dotted line represents the time course of oocyte maturation as determined by percentage of GVBD. Asterisks denote statistical significance by two-way ANOVA, followed by Bonferroni posttest, where *** is P < 0.001, and ** is P < 0.01. Each point is the mean ± SEM of three independent experiments conducted on different days. B) Groups of 50 oocytes were dissected from eCG-primed immature mice 2 h after injection of vehicle or 5 IU of hCG. For the cGMP assay, oocytes were processed as detailed in Materials and Methods, and the cGMP content was measured by ELISA with acetylated samples. The asterisk indicates statistical difference, where P < 0.05.

FIG. 4.

Activation of the EGF network is required for the LH-induced drop in follicular cGMP. Groups of 10–20 POFs were cultured with the specified treatments for the indicated times, and cGMP levels were determined as described in Materials and Methods. Different letters at the top of the bars indicate statistical difference, where P < 0.05. A) POFs were preincubated in the presence or absence of 500 nM AG1478 and then stimulated for 2 h with either 5 IU or rLH. B) POFs were treated with or without 100 nM AREG for 2 h. C) POFs were cultured in the presence or absence of 100 nM AREG for the indicated times. Data are expressed as percentage of cGMP per follicle compared with the control for the indicated time. The data represent the mean ± range of two independent experiments.

FIG. 5.

Luteinizing hormone decreases cGMP content in cultured follicles, while sildenafil and DEA increase cGMP. Groups of 20–30 POFs were incubated in the presence or absence of rLH for 1 h with or without preincubation with 1 μM sildenafil (SIL) and 10 μM DEA for a total culture time of 2 h. Cyclic GMP levels were determined as described in Materials and Methods. Data shown are femtomoles of cGMP per follicle ± SEM of at least three independent experiments. Different letters at the top of the bars indicate statistical difference, where P < 0.05.

FIG. 6.

In cultured follicles, PDE5 inhibitors prevent LH-induced maturation, and DEA augments this effect. Groups of 10–20 POFs were used for each experimental condition. A) Follicles were preincubated with or without 1 μM sildenafil (SIL) for 1 h, followed by 10 μM DEA for 5 min when indicated. POFs were stimulated with 5 IU of rLH for 4 h; follicles were then punctured, and oocytes were scored for signs of meiotic resumption (GVBD). B) Follicles were preincubated in the presence of 1 μM tadalafil (TADA) for 1.5 h and then stimulated with rLH. C) Follicles were preincubated for 1.5 h in the presence of 1 μM tadalafil with or without LH stimulation, and meiotic resumption was scored at the end of overnight culture. The graphs show the mean ± SEM of at least three independent experiments. Different letters at the top of the bars indicate statistical difference, where P < 0.01.

FIG. 7.

Sildenafil and DEA do not affect spontaneous maturation in mouse COCs and DOs. A) Groups of 20–30 COCs were incubated in 100-μl drops of media under mineral oil for 2.5 h in the presence or absence of 1 μM sildenafil (SIL) alone or with 10 μM DEA plus sildenafil. The maturation stage of oocytes was scored after removal of the granulosa cells and is expressed as percentage of GVBD. B) The DOs were collected in media with or without 1 μM sildenafil and were placed in 100-μl drops of media under mineral oil. The DOs were scored for meiotic maturation as described in Materials and Methods, with Time 0 representing the time of DO isolation. The data represent the mean ± SEM of five independent experiments for A and two independent experiments for B.

FIG. 8.

A pool of cGMP in the oocyte contributes to meiotic arrest through PDE3A. A) Wild-type DOs were microinjected in the presence of 3.5 mM HX with inactive PDE5 or active PDE5 or with active PDE5 preincubated with 1 μM sildenafil (SIL) for 1 h. B) Pde3a−/− oocytes were injected in the absence of HX with inactive PDE5, active PDE5, or PKI-tide. For both experiments, GVBD was scored 2.5 h after injection. The number of injected oocytes that underwent meiotic resumption out of the total number of injected oocytes are listed at the top of each bar. The graphs show the mean ± SEM of at least two independent experiments. Different letters at the top of the bars indicate statistical difference, where P < 0.05.