Hormonal coordination of natriuretic peptide type C and natriuretic peptide receptor 3 expression in mouse granulosa cells

Abstract

Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA, and subsequent human chorionic gonadotropin (hCG) treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with equine chorionic gonadotropin to further define the mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by estradiol (E2), with augmentation by follicle-stimulating hormone (FSH), but FSH or luteinizing hormone (LH) alone had no effect. Thus, estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. Human chorionic gonadotropin increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.

Figures

FIG. 1

Expression of Nppc mRNA in cumulus cells and MGCs in vivo from mice stimulated with eCG and hCG. Equine chorionic gonadotropin was administered to 21-day-old mice for periods up to 48 h, and hCG was administered 44 h later. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 2

Levels of Nppc mRNA in cultured MGCs. Cells were collected from mice not stimulated with eCG and then cultured for up to 24 h without hormonal treatment. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 3

Effect of FSH (F, 5 ng/ml), LH (L, 10 ng/ml), and 17β-estradiol (E, 100 nM) on levels of (A) Nppc and (B) Npr2 mRNA in cultured MGCs. MGCs were collected from mice not stimulated with eCG and cultured for 24 h with various hormones after the initial culture for 24 h without hormones to allow for attachment of viable cells. C, control value, when no hormones were added during culture. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 4

Effect of oocytes on expression of Nppc and Lhcgr mRNA in cumulus-oocyte complexes (COC) and oocytectomized (OOX) cumulus in vitro. Complexes were collected from mice stimulated with eCG and then cultured for 24 h with combination of E2 plus FSH and 10 μM milrinone to maintain oocytes at the GV stage. OOX (oocytectomized) complexes were cocultured with 0–5 oocytes/μl. COC, intact cumulus-oocyte complexes. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 5

Effect of LH and EGF on levels of Nppc mRNA in cultured MGCs. MGCs were collected from mice not stimulated with eCG, cultured for 24 h without hormones to allow attachment of viable cells, then cultured for 24 h with E2 and FSH. A) MGCs were cultured for an additional 6 h with LH (1 μg/ml) or EGF (10 ng/ml). B) MGCs were cultured for an additional 2–6 h with EGF. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 6

Effect of LH on levels of Areg (blue line) and Ereg (red line) mRNA in cultured MGCs. MGCs were collected from mice not stimulated with eCG, cultured for 24 h without hormones to allow attachment of viable cells, then cultured for 24 h with E2 and FSH. LH (1 μg/ml) was then added and samples taken at time 0 and at six 1-h intervals thereafter. The relative values for each time point were normalized and compared to the 0 h point. Upper case letters are used for statistical comparison of Areg values versus 0 h and lower case for Ereg values. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 7

Effect of LH and EGF on levels of cGMP in cultured MGCs. MGCs were collected from mice not stimulated with eCG, cultured for 24 h without hormones to allow attachment of viable cells, then cultured for 24 h with E2 and FSH. They were then cultured for an additional 2 h with LH (1 μg/ml) or EGF (10 ng/ml). Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 8

Expression of Npr3 mRNA in cumulus cells and MGCs in vivo from mice stimulated with eCG and hCG. Equine chorionic gonadotropin was administered to 21-day-old mice for periods up to 48 h and hCG was administered 44 h later. A) In situ hybridization showing localization of Npr3 mRNA expression. Transcripts were not detected in cumulus cells (cc), but were observed in MGCs 3 h post-hCG. Upper and lower panels indicate bright-field and dark-field images, respectively. OO indicates oocyte. Bars = 200 μm. B) Levels of Npr3 mRNA in cumulus cells and MGCs by qRT-PCR. Relative expression levels not indicated by the same letter are significantly different (P < 0.05).

FIG. 9

Effect of LH, EGF, and the EGF receptor kinase inhibitor (AG1478) on levels of Npr3 mRNA in cultured MGCs. LH (1 μg/ml, red) or EGF (10 ng/ml, blue) were added to cultures of MGCs after 24 h treatment with E2 plus FSH. Samples were taken at the times shown and relative levels of Npr3 mRNA determined by qRT-PCR. The EGFR kinase inhibitor AG1478 (1 μM) was added at the same time as either LH or EGF. Samples treated with AG1478 are shown with dotted lines. All the data are expressed as a fold difference from time 0. Each point represents the mean ± SEM of three independent experiments.

FIG. 10

NPPC levels in Npr3lgj/Npr3lgj mutant and heterozygotes (het) at various times after administration of 5 IU hCG to eCG-stimulated mice. Bars indicate the mean ± SEM. The numbers over the bars indicate the number of ovaries in which the NPPC content was measured. ns, no significant difference.