Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes

Abstract

Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte-follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9-bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself.

Fig. 1.

Abbreviated pathway to illustrate cGMP production and its role in oocyte meiotic arrest. The action of IMPDH, which converts IMP to GMP, is blocked by mizoribine. However, the inhibitory effect of mizoribine can be bypassed by addition of exogenous guanosine downstream of IMPDH inhibition. NPR2 is a guanylyl cyclase activated by the ligand NPPC and produces cGMP from GTP. cGMP enters the oocyte via gap junctions, represented by the heavy line at the point of entry of cGMP to the oocyte. Within the oocyte, cGMP inhibits PDE3A, a cAMP phosphodiesterase, which, if uninhibited, would degrade oocyte cAMP and cause the resumption of meiosis. IMP can be produced either by de novo pathways or by salvage of hypoxanthine. However, as indicated in the text, little salvaged hypoxanthine is converted to guanylyl metabolites in COCs (22, 23) but rather may promote the de novo synthesis of IMP or prevent its loss from availability for conversion to GMP by IMPDH.

Fig. 2.

Effect of NPPC and hypoxanthine on oocyte maturation in vitro. COCs were cultured for 24 h in medium containing NPPC (0–25 nM) and/or HX (0–1 mM), and the percentage of oocytes that underwent GVB was determined. All media contained 100 µM E2 to maintain NPR2 receptors throughout the culture period (35). Bars indicate the mean ± SEM of seven experiments. Bars without letters in common were considered significantly different (P < 0.05).

Fig. 3.

Effect of NPPC and/or HX on maintenance of meiotic arrest (Left) and cGMP levels (Right). COCs were cultured for 24 h in medium containing neither NPPC nor HX (control), or 10 nM NPPC, 4 mM HX, or both (blue bars). The COCs cultured with NPPC or HX, or both, were also treated with 200 µM mizoribine (red bars) or mizoribine plus 50 µM guanosine (yellow bars). Mizoribine treatment prevented the GVB-inhibiting effects of NPPC, HX, or both, and this inhibition was circumvented by guanosine (Left). NPPC and NPPC+HX promoted dramatic increases in the levels of cGMP in the COCs (Right). These increases were blocked by mizoribine, and the effect of mizoribine was bypassed by exogenous guanosine. Interestingly, HX did not raise the level of cGMP above that in control COCs. However, this level was decreased by mizoribine treatment. Thus, although HX maintained meiotic arrest (Left), an increase in cGMP was not detected (Right). Bars indicate the mean ± SEM of four experiments. Bars without letters in common were considered significantly different (P < 0.05).

Fig. 4.

Effect of ODPFs on levels of Impdh1 and Impdh2 mRNA and IMPDH2 protein in cumulus cells. Oocytectomized cumulus cells (OOX) were cultured alone or cocultured with oocytes (0.5–2.0 oocytes per µL) and levels of Impdh1 and Impdh2 transcripts (A) and IMPDH protein (B) were assessed after a 24-h culture. Intact COCs were also cultured for comparison. OOX caused a dramatic reduction in mRNA and protein levels. ODPFs produced by 0.5 oocytes per µL were sufficient to sustain both transcript and protein at the same or above the levels of intact COCs. Bars indicate the mean ± SEM of four experiments. Bars without letters in common were considered significantly different (P < 0.05).

Fig. 5.

Effect of ODPFs on cGMP levels in OOX cumulus cells. (A) OOX cumulus cells were cultured alone or cocultured with oocytes (0.5–2.0 oocytes per µL) for 24 h and levels of cGMP were determined. OOX caused a dramatic reduction in cGMP levels, but these levels were mantained by coculture with oocytes. (B) The effects of recombinant mouse ODPFs on cGMP levels. OOX cumulus cells were cultured without ligands (OOX) or with mouse GDF9 (30 ng/mL), BMP15 (30 ng/mL), BMP15+GDF9 (both at 30 ng/mL), or GDF9–BMP15 heterodimer (3 ng/mL). These were maximally effective concentrations needed for cumulus expansion or the expression of expansion-related transcripts when used in concert with EGF in previous studies (33). The levels of cGMP were determined after a 24-h culture. Bars indicate the mean ± SEM of four experiments. Bars without letters in common were considered significantly different (P < 0.05).

Fig. 6.

Working model of the bidirectional mechanism required for the maintenance of meiotic arrest. Although it has been widely accepted for many years that follicular granulosa cells maintain oocytes in meiotic arrest before the preovulatory surge of LH, this model illustrates that oocytes themselves orchestrate this action of granulosa cells. ODPFs control the activity of IMPDH (green arrow). This enzyme provides the substrate for NPR2, a guanylyl cyclase that converts GTP to cGMP. The levels of cGMP in cumulus cells are elevated by ODPFs (green arrow). The purine hypoxanthine also requires IMPDH activity to participate in meiotic arrest in vitro. However, if hypoxanthine participates in meiotic arrest in vivo, it may enhance the cAMP phosphodiesterase-inhibiting activity of cGMP within the oocyte. Cumulus cells take up hypoxanthine from the follicular fluid. Both cGMP and hypoxanthine diffuse into the oocyte from cumulus cells via gap junctions represented by the heavier lines at their points of entry into the oocyte. Disruption of this arresting system would result in activation of PDE3A, and the oocytes would resume meiosis.