Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes

Abstract

Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents their precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C (Nppc) messenger RNA (mRNA), whereas cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC increased cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.

Figures

Fig. 1

Expression of Nppc and Npr2 mRNA by granulosa cells. (1A) In situ hybridization showing localization of Nppc and Npr2 mRNA expression in prepubertal mouse ovaries 48 hr after eCG injection. Histology is shown in bright field and localization of specific mRNAs is shown in dark field images. * indicates mural granulosa cells; arrows indicate cumulus cells. Bar = 200 μm. (1B) Comparison of steady-state levels of Nppc and Npr2 mRNAs by mural and cumulus cells by QRT-PCR. Levels of transcripts in cumulus cells are expressed relative to that measured in mural granulosa cells, mean of 3 experiments was set to a value of 1. *** indicates P < 0.001; * indicates P < 0.05. (1C) Effect of oocytes on Npr2 expression in cumulus cells. Levels of Npr2 mRNA in cumulus cells cultured as intact COCs (COC), oocytectomized (OOX) cumulus cells, or OOX cumulus cells co-cultured with fully-grown oocytes (OOX+OO; 2 oocytes/μl) for 14 hr were determined. The mean value in COC group was set as 1. (1D) Effect of GDF9, BMP15, and FGF8 on Npr2 mRNA levels in OOX cumulus cells. OOX cumulus cells were co-cultured with either denuded oocytes (Oocyte) or 500 ng/ml mouse GDF9, 500 ng/ml human BMP15, 100 ng/ml mouse FGF8B, or the various combinations indicated for 14 hr and levels of Npr2 mRNA determined. The mean value in the control (no-treatment) group was set as 1. Values indicated by different letters are significantly different (P < 0.05).

Fig. 2

Effect of natriuretic peptides on gonadotopin-independent (spontaneous) resumption of meiosis and production of cAMP and cGMP in vitro (2A) Effect of natriuretic peptides on spontaneous maturation of cumulus cell-enclosed oocytes in vitro. Oocytes were cultured 4 hr in medium containing human NPPA, NPPB, or NPPC and assessed for germinal vesicle breakdown (GVBD) indicative of the resumption of meiosis. NPPC dose-dependently inhibited resumption of cumulus cell-enclosed oocytes with a maximum effect at 100 nM. Neither NPPA or NPPB had detectable activity in preventing GVBD at 100 nM. Effect of natriuretic peptides on levels of cGMP (2B) and cAMP (2C) in cumulus and oocyte compartments of cumulus-oocyte complexes (COCs). COCs were incubated in medium containing 100 nM human NPPA, NPPB or NPPC for 1 hr, and levels of cGMP and cAMP in oocytes and cumulus cells were evaluated by ELISA. NPPC, but not NPPA or NPPB, promoted increased cGMP levels in both oocytes and cumulus cells but an increase in cAMP was seen only in oocytes. CC indicates the total cumulus cells surrounding one oocyte. (*P < 0.05 compared with control).

Fig. 3

Failure to maintain meiotic arrest in Graafian (late antral) follicles of Npr2cn-2J/Npr2cn-2J and Nppclbab/Nppclbab mutant mouse ovaries. (A) A prophase-arrested oocyte (GV indicates germinal vesicle) within a late antral follicle of a control Npr2wt/Npr2? ovary. (B) An oocyte with metaphase I (MI) chromosomes within a late antral follicle of an Npr2cn-2J/Npr2cn-2J mutant ovary. (C) Percentages of oocytes that had resumed meiosis, counted in sections of ovaries from control Npr2wt/Npr2? and mutant Npr2cn-2J/Npr2cn-2J mice that were treated with eCG for 44 hr. Graph shows mean ± SEM of 6 ovaries; numbers above bars indicate number of follicles examined. (D-E) Ovarian follicles after development as grafts under kidney capsules of immunodeficient hosts for 30 days. (D) A prophase-arrested oocyte within a late antral follicle of a heterozygous control Nppcwt/Nppclbab ovary. (E) An oocyte with metaphase II (MII) chromosomes and a polar body, within a late antral follicle of a mutant Nppclbab/Nppclbab ovary. (F) Percentages of oocytes that had resumed meiosis, counted in sections of Nppcwt/Nppclbab and Nppclbab/Nppclbab grafted ovaries. Graph shows mean ± SEM of 6 ovaries; numbers above bars indicate number of follicles examined. The terms “Early” and “Late” antral follicles are used as defined by Pedersen and Peters (15). Bars = 100 μm.

Fig. 4

Model depicting the role of NPPC and NPR2 and oocyte-derived paracrine factors (ODPF) in the maintenance of oocyte meiotic arrest. See text for details. ADCY, adenylyl cyclase; GPR3/12, G-protein coupled receptors 3 and 12; Gs, GNAS (guanine nucleotide binding protein, alpha stimulating) complex; ODPF, oocyte-derived paracrine factors.