Skip to main content
NCBI home page
Biology of Reproduction logoLink to Biology of Reproduction
. 2012 Dec 19;88(2):42. doi: 10.1095/biolreprod.112.104810

Hormonal Coordination of Natriuretic Peptide Type C and Natriuretic Peptide Receptor 3 Expression in Mouse Granulosa Cells1

Kyung-Bon Lee 3, Meijia Zhang 4, Koji Sugiura 5, Karen Wigglesworth 3, Tracy Uliasz 6, Laurinda A Jaffe 6, John J Eppig 3,2
PMCID: PMC3589232  PMID: 23255339

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.

Keywords: epidermal growth factor, estradiol/estradiol receptor, estrogens, follicle-stimulating hormone, gonadotropins, granulosa cells, luteinizing hormone, natriuretic peptide receptor 3, natriuretic peptide type C

Expression of natriuretic peptide type C (NPPC) in mouse granulosa cells is regulated by estrogen, gonadotropins, and EGF, but ovarian NPPC peptide levels are unaffected by mutation of the NPR3 clearance receptor.

INTRODUCTION

Oocytes in antral stage follicles are maintained in meiotic arrest, at the germinal vesicle (GV) stage, until the preovulatory surge of luteinizing hormone (LH). The LH signal is amplified by promoting the production of EGF-like peptides first by mural granulosa cells (MGCs), which line the follicle wall, and then by cumulus cells, which are closely associated with oocytes [1–6]. Maintenance of meiotic arrest in these follicles requires natriuretic peptide type C (NPPC, also known as C-type natriuretic peptide), produced mainly by MGCs, and the NPPC cognate receptor, natriuretic peptide receptor 2 (NPR2), which is expressed most highly by cumulus cells, but also by MGCs in an outwardly decreasing gradient [7–9]. The binding of NPPC to NPR2, a transmembrane guanylyl cyclase, results in granulosa cell production of cyclic guanosine monophosphate (cGMP) that is transferred via gap junctions to the oocyte [7, 10]. In oocytes, cGMP inhibits phosphodiesterase 3A (PDE3A), thereby preventing cyclic adenosine monophosphate (cAMP) degradation and maintaining oocyte meiotic arrest [11]. Thus, both Nppc and Npr2 expression are crucial for maintaining meiotic arrest in the mouse.

Levels of Nppc and Npr2 mRNA are increased in MGCs and cumulus cells in vivo by stimulation of follicular development with equine chorionic gonadotropin (eCG) [7–9]. However, in vitro, Npr2 mRNA levels in cumulus cells, the ability of these cells to respond to NPPC by cGMP production, and maintenance of oocyte meiotic arrest were promoted by 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), while follicle-stimulating hormone (FSH) alone had no effect [8].

Stimulation of LH receptors with either LH or human chorionic gonadotropin (hCG) decreases cGMP in granulosa cells, thus releasing the cGMP-mediated inhibition of meiotic arrest [10, 11]. The cGMP decrease results at least in part from a decrease in guanylyl cyclase activity in both the mural granulosa and cumulus cells that occurs even in the presence of saturating concentrations of NPPC [12]. In addition, LH receptor stimulation causes a decrease in NPPC in the ovary that is likely to further decrease guanylyl cyclase activity [9, 12]. These consequences of LH receptor stimulation are integral components of a complex network of processes participating in the physiological induction of oocyte meiotic resumption in mice. Given the crucial role of NPPC in maintaining meiotic arrest and participating in other aspects of follicular development [7, 13–16], this study focuses on the regulation of Nppc expression in MGCs and cumulus cells by hormones and oocyte-derived paracrine factors in culture.

Levels of Npr2 mRNA are higher in cumulus cells than in MGCs, and ODPFs promote this expression in their neighboring granulosa cells [7]. In contrast, Nppc mRNA expression is higher in MGCs than cumulus cells [7]. Similarly, levels of Lhcgr, Cyp11a1, and Cd34 mRNAs are higher in MGCs than cumulus cells, and expression of these transcripts is suppressed in cumulus cells by ODPFs, thus explaining the differential expression in MGCs versus cumulus cells [17]. Therefore, this study tests the hypothesis that levels of Nppc mRNA are higher in MGCs than cumulus cells because of the suppression of expression in cumulus cells by ODPFs.

Finally, the regulation of natriuretic peptide receptor 3 (Npr3) mRNA levels and the possible role of NPR3 in regulating NPPC levels in ovaries were assessed. This is of potential interest because NPR3, which has no guanylyl cyclase domain and is considered to be a clearance receptor, can bind and internalize NPPC for degradation [18] and could, therefore, have a function in controlling ovarian NPPC levels and oocyte meiotic maturation. Npr3longjohn (lgj) is an in-frame 36-bp deletion affecting the extracellular NPPC-binding domain of NPR3, and homozygous Npr3lgj/Npr3lgj mutant mice exhibit skeletal abnormalities demonstrating a phenotypic consequence of NPR3 modification [19]. We hypothesize that the clearance receptor NPR3 functions to decrease NPPC levels in ovarian follicles in response to stimulation of the LH receptor and promote meiotic resumption.

MATERIALS AND METHODS

Mice

Female B6SJLF1 mice and Npr3lgj/Npr3lgj mutant mice, initially obtained from The Jackson Laboratory (stock number 003506), and controls were produced and raised in the research colony of the investigators at The Jackson Laboratory and were used for all the experiments. Ovaries were collected from 18- to 22-day-old females with or without hormonal stimulation. Animals were maintained according to the Guide for the Care and Use of Laboratory Animals (Institute for Learning and Animal Research), and the protocols were approved by the Jackson Laboratory Animal Care and Use Committee.

Chemicals and Hormones

All the chemicals were purchased from Sigma-Aldrich unless otherwise stated. Human recombinant FSH and recombinant human LH were obtained from the EMD-Serono Research Institute.

Isolation of MGCs and Cumulus Oocyte Complexes

MGCs were collected by gentle puncture with a 25 gauge syringe needle of antral follicles from mice that were not stimulated with eCG. Cumulus-oocyte complexes (COCs), with the oocytes at the GV stage, were collected from mice 48 h after stimulation with eCG. To examine Npr3 mRNA expression levels in vivo, MGCs and cumulus cells were isolated at various times from 21-day-old mice that were stimulated with 5 international units (IU) of eCG (for periods up to 48 h) or eCG followed with 5 IU hCG 44 h later.

Culture of MGCs and COCs

Studies of the hormonal control of Nppc mRNA and cGMP levels were carried out using either monolayer cultures of MGCs or isolated COCs. The medium used for these studies was bicarbonate-buffered minimum Eagle medium (MEM)-alpha with Earle balanced salts (M0894) supplemented with 75 μg/ml penicillin G, 50 μg/ml streptomycin sulfate, and 3 mg/ml bovine serum albumin. In order to promote the attachment of granulosa cells in the absence of serum, 48-well tissue culture dishes were first coated with 20 μg/ml entactin-collagen IV-laminin (ECL) extracellular matrix (Millipore) for at least 1 h at 37°C as described by the supplier before adding MGCs. The ECL-coated wells were then washed with MEM-alpha including 3 mg/ml bovine serum albumin. MGCs were cultured on this matrix for 24 h without hormones, and the unattached nonviable MGCs were removed by washing with culture medium at the end of this initial culture. The attached cells were cultured with or without hormones for various times. All the cultures were maintained at 37°C in a modular incubation chamber (Billups Rothenberg) in an atmosphere of 5% O2, 5% CO2, and 90% N2.

Microsurgical Removal of Oocytes from COCs

Oocytes were microsurgically removed (oocytectomy, OOX) from COCs isolated from the ovaries of 18- to 22-day-old mice 44–48 h after injection of 5 IU eCG. The procedures for OOX and subsequent coculture with or without GV stage denuded oocytes were carried out as described previously [20].

RNA Isolation and Quantitative RT-PCR

After harvesting, cells were stored at −80°C until analyzed for relative expression levels of mRNA. Total RNA was isolated from frozen samples using the RNeasy micro-RNA isolation kit (Qiagen) as recommended by the manufacturer. Reverse transcription was performed directly after RNA isolation using the QuantiTect reverse transcription kit (Qiagen) as recommended by the manufacturer. Reverse transcribed cDNA was diluted 1:5 with nuclease-free water and stored at −80°C until quantitative real-time PCR (qRT-PCR) was performed. The relative amount of target gene expression for each sample was conducted using an ABI 7500 real-time PCR instrument (Applied Biosystems). PCR primer sequences for Nppc, Npr2, Areg, Ereg, and Rpl19 were reported previously [7, 21]. The Npr3 primer sequences were as follows: 5′–GTTCCAAATGCGATCGAATGT-3′ for forward and 5′-CCCACAACGATTCCTGTCACT-3′ for reverse. Levels of mRNA were normalized relative to abundance of an endogenous control housekeeping transcript: ribosomal protein L 19 (Rpl19). Relative levels of target gene expression for each sample were calculated using the formula 2−ΔΔCt as described previously [22]. The identities of all amplicons were validated by sequencing the purified PCR products.

Measurement of cGMP and Genomic DNA Levels

For measurement of cGMP, cells were removed from the monolayers using MEM-alpha medium containing 0.2 mM 3-isobutyl-1-methylxanthine and 0.05% trypsin. Each sample was divided in half with one half used for measurement of genomic DNA content and the other for cGMP assay. For the cGMP assay, cells were washed with Dulbecco PBS and solubilized in 100 μl of 0.1 M HCl on ice for at least 10 min before storage at −80°C until assay. Frozen samples were thawed and centrifuged at 12 000 × g for 5 min, and the supernatants were then transferred to a clean microcentrifuge tube and dried at 60°C using miVac (Genevac). The samples were resuspended in 100 μl of enzyme immunoassay (EIA) buffer and cGMP was measured using a cGMP-EIA kit (Cayman Chemical Company), a Spectra Max 250 plate reader (Molecular Devices), and a spreadsheet tool for data analysis from the Cayman website (http://www.caymanchem.com/analysis/eia) [7]. The cGMP data were normalized to the total DNA in the sample. Genomic DNA was isolated using the Wizard Genomic Purification kit (Promega), and its concentration was determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific).

In Situ Hybridization of Npr3 mRNA

Localization of Npr3 mRNA in the ovary was determined by in situ hybridization as previously described [7, 23]. Briefly, ovaries isolated from 21-day-old mice that were stimulated with eCG (48 h) or eCG (48 h) followed by hCG (3 h) were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 7 μm onto Superfrost plus microscope slides (Fisher Scientific) for in situ hybridization.

Measurement of Ovarian NPPC Levels in Control and Npr3lgj/Npr3lgj Mutant Mice

At various times after administration of 5 IU hCG to eCG-stimulated mice, ovaries were removed from Npr3lgj/Npr3lgj mutant mice and heterozygotes, and NPPC levels were determined by an ELISA method as described previously [12].

Statistical Analysis

JMP10 software was used to conduct the Student t-test or the Tukey-Kramer honestly significant difference test for paired or multiple comparisons, respectively (SAS Institute, Inc.); P < 0.05 is considered to be significantly different. All the experiments were performed independently at least three times. Within each experiment, duplicate measurements are made for each group. Data are presented as mean ± SEM.

RESULTS

Nppc mRNA Levels in MGCs and Cumulus Cells In Vivo

Nppc mRNA levels in MGCs increased after injection of mice with eCG approximately 4-fold within 24 h and further by 48 h (Fig. 1). The levels decreased significantly in eCG-stimulated MGCs by 2 h post-hCG and declined to pre-eCG levels by 3 h post-hCG, as shown by Kawamura et al. [9] in whole ovaries (Fig. 1). In contrast, eCG promoted no significant increase in Nppc mRNA levels in cumulus cells. Nevertheless, Nppc mRNA in these cells decreased after administration of hCG, to a level about half that in cumulus cells pre-eCG (Fig. 1). Thus, hCG treatment of eCG-stimulated mice decreased Nppc mRNA levels in both MGCs and cumulus cells.

FIG. 1.

FIG. 1

Open in a new tab

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).

Nppc mRNA Levels in MGCs In Vitro

In order to investigate the hormonal regulation of Nppc mRNA levels in MGCs, a system for in vitro analysis was established. Initial experiments in vitro were conducted using small clumps and single MGCs collected after puncturing follicles with needles and then gently dispersed by pipetting. Thus, MGCs were not attached to an extracellular matrix as most of them would be in vivo. Dramatically lower levels of Nppc mRNA levels were found 1 h after isolation, and the levels continued to decrease for 8 h (Fig. 2). The decreasing levels of Nppc transcripts probably resulted from damaged and nonviable cells as well as loss of expression by viable cells in the absence of hormonal stimulation in vivo. Therefore, subsequent experiments testing the effects of various hormones and growth factors were conducted using only MGCs that became attached to an ECL matrix, in the absence of serum, during the first 24 h of culture; the nonviable unattached cells were discarded. There was no morphological indication of luteinization observed during 48 h of culture.

FIG. 2.

FIG. 2

Open in a new tab

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).

Effect of Hormonal Treatments on Relative Levels of Nppc and Npr2 mRNA Levels in MGCs In Vitro

Various hormones were added to the monolayer cultures for 24 h after removal of nonattached MGCs. Because eCG, which exhibits both FSH and LH activity [24, 25], promoted elevated Nppc mRNA levels in vivo, it was anticipated that FSH, LH, or the combination of both might increase Nppc mRNA levels in vitro. However, neither FSH (5 ng/ml) nor LH (10 ng/ml), alone or together, promoted elevated transcript levels (Fig. 3A). Nevertheless, E2 (100 nM) raised the levels of Nppc mRNA in cultured MGCs approximately 10-fold, and this was increased even further, to about 20-fold relative to the control, by the combination of E2 plus FSH, but not by E2 plus LH (Fig. 3A). The highest levels, an increase of about 30-fold relative to the control, were promoted by the combination of E2 plus FSH and LH.

FIG. 3.

FIG. 3

Open in a new tab

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).

To test the possibility that eCG could stimulate theca-derived paracrine factors to promote Nppc mRNA expression by MGCs, cells were cultured with hepatocyte growth factor or fibroblast growth factor 7 (also known as keratinocyte growth factor), which are produced by mesenchyme-derived theca cells and regulate growth and steroidogenesis by granulosa cells [26]. However, neither of these paracrine factors, nor testosterone nor dihydrotestosterone, affected Nppc mRNA expression by MGCs (data not shown).

As seen for Nppc, neither FSH nor LH, alone or together, promoted elevation of Npr2 transcript levels in MGCs in vitro. However, levels were increased about 3-fold by E2 alone, though, unlike the regulation of levels of Nppc transcripts in MGCs, the combination of E2 plus FSH did not increase levels above that of E2 alone (Fig. 3B).

Effect of Oocytes on Nppc mRNA Expression by Cumulus Cells

Fully grown oocytes profoundly affect the patterns of gene expression by granulosa cells [17, 27, 28]. Cumulus cells and periantral MGCs express Npr2 mRNA at levels that are higher than in the MGCs that line the follicular wall [7, 8]. Because ODPFs promote the expression of Npr2 mRNA by cumulus cells, the differential expression of this transcript is probably due to the stimulatory effect of the ODPFs [7]. Conversely, expression of luteinizing hormone choriogonadotropin receptor (Lhcgr) mRNA is much greater by MGCs than by cumulus cells, and ODPFs suppress expression by cumulus cells [17, 29]. Because expression of Nppc mRNA is much greater in MGCs than in cumulus cells, we hypothesized that ODPFs suppress Nppc mRNA expression in cumulus cells. To test this, we measured Nppc mRNA levels in intact COCs, OOX cumulus cells, and OOX cumulus cells cocultured with 0, 0.5, 2.0, or 5.0 cumulus cell-denuded oocytes/μl; all the groups were cultured in medium supplemented with E2 plus FSH and 10 μM milrinone to maintain oocytes at the GV stage. There was an oocyte dose-dependent suppression of Lhcgr mRNA as expected on the basis of previous studies [17, 29]. However, in contrast to our hypothesis, OOX resulted in decreased Nppc mRNA levels that were restored to the levels of cumulus cells in intact COCs by coculture with oocytes (Fig. 4). Thus, unlike expression of Lhcgr mRNA, Nppc mRNA levels were not suppressed by ODPFs but rather stimulated in vitro, despite the fact that Nppc mRNA is normally expressed at much lower levels in cumulus cells than in MGCs in vivo.

FIG. 4.

FIG. 4

Open in a new tab

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).

Effect of LH or EGF on Nppc mRNA and cGMP Levels in Cultured Granulosa Cells

Because hCG-treatment of eCG-stimulated follicles induced a rapid decrease in Nppc mRNA levels in ovaries [9] and MGCs and because this stimulation of LH receptors is known to promote maturational changes amplified by EGF-like peptides [1–6], the effects of LH and EGF on expression of Nppc transcript levels in cultured MGCs were determined. MGCs were cultured in medium without hormonal supplementation for 24 h to allow attachment of viable cells to ECL as described above. After removal of unattached cells, the MGCs were cultured for an additional 24 h in medium containing E2 plus FSH, which, as already described, promoted elevated levels of Nppc transcripts. Then, either LH (1 μg/ml) or EGF (10 ng/ml) was added for 6 h, while a control culture had no further hormonal additions. Under these conditions, LH did not decrease Nppc mRNA levels, but EGF did (Fig. 5A). The decrease in Nppc transcript levels occurred within 2 h of adding EGF to the cultures (Fig. 5B). Expression of both Areg and Ereg transcripts increased within 1 h of LH stimulation, reached peak levels by 3 h, and decreased almost to basal levels by 6 h post-LH (Fig. 6). However, the increase in Areg mRNA was of much greater magnitude than that of Ereg at 3 h: 32-fold versus 6-fold, respectively. Thus, production of EGF-like peptides probably begins within an hour of LH-treatment in vitro. The inability of LH to decrease Nppc RNA in vitro could be explained by the dilution of these peptides in the culture medium despite using the minimum possible volumes of medium.

FIG. 5.

FIG. 5

Open in a new tab

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.

FIG. 6

Open in a new tab

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).

Oocyte meiotic arrest depends upon NPPC-mediated generation of cGMP by granulosa cells and subsequent transfer of cGMP to oocytes [7, 10]. Granulosa cell cGMP levels decrease after stimulation of LH-receptors in intact follicles [10, 11, 30], and a transient decrease has also been seen in rat granulosa cells in culture [31]. Therefore, the effect of LH or EGF on cGMP levels in MGCs cultured as described here was determined. MGCs cultures were stimulated for 24 h with E2 plus FSH and then addition of LH or EGF for 2 h. As observed for Nppc mRNA, cGMP levels were decreased by EGF treatment, but not by LH (Fig. 7). Thus, stimulation of the EGF receptor contributes to the decrease in Nppc mRNA and cGMP levels in cultured MGCs.

FIG. 7.

FIG. 7

Open in a new tab

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).

Hormonal Control of Npr3 mRNA Levels In Vivo and In Vitro

Because NPR3 is an NPPC clearance receptor [18], it is a good candidate to play a role in the post-LH decrease in ovarian NPPC levels that have been reported elsewhere [9, 12]. Therefore, the regulation of Npr3 mRNA was assessed both in vivo and in vitro. In situ hybridization shows very low levels of follicular Npr3 mRNA after stimulation of mice with eCG. However, expression increases dramatically in MGCs, but not cumulus cells, by 3 h after administration of hCG (Fig. 8A). Quantitative RT-PCR showed an ∼35-fold increase in MGCs at 1 h after administration of hCG and an ∼500-fold increase at 3 h, relative to levels in MGCs from animals not treated with hCG. Although levels of Npr3 mRNA also increase after hCG in cumulus cells, the levels achieved are far lower than measured in MGCs (Fig. 8B). Thus, eCG elevates Nppc and Npr2 mRNA in MGCs in vivo, and these levels are reduced after hCG administration. However, the effects on Npr3 mRNA are the opposite: eCG treatment does not increase Npr3 mRNA levels in MGCs, but hCG treatment does.

FIG. 8.

FIG. 8

Open in a new tab

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).

Using the same culture system as described above, MGCs were cultured for 24 h in medium containing E2 plus FSH, and then LH (1 μg/ml) or EGF (10 ng/ml) was added and samples were taken 0, 1, 3, and 5 h later. Although the levels of Npr3 mRNA increased in both groups after 3 h, the highest level, an ∼6-fold increase, was observed in the group treated with LH (Fig. 9). No additional increase was seen when EGF and LH were applied together (data not shown). An inhibitor of EGF-receptor kinase, AG1478 (1 μM), had no effect on LH-stimulated Npr3 mRNA expression, but, as expected, it prevented the EGF-stimulated increase (Fig. 9). Thus, LH-stimulation of Npr3 mRNA synthesis in cultured MGCs appears to be independent of activation of the EGF receptor and is therefore probably not mediated or amplified by production of EGF-like peptides, though other factors promoted by hCG/LH probably amplify Npr3 expression in vivo.

FIG. 9.

FIG. 9

Open in a new tab

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.

Effect of Npr3 Mutation on Oocyte Development and Ovarian NPPC Levels

Because a decrease in ovarian NPPC levels has been correlated with the resumption of meiosis in oocytes [9, 12] and because NPR3 can reduce NPPC levels [18], we determined the effect of an Npr3 mutation [19] on the kinetics of hCG-induced oocyte maturation and reduction in NPPC levels. Twenty-day-old heterozygous and Npr3lgj/Npr3lgj mutant mice were injected with eCG and 44 h later with hCG to induce meiotic resumption. Ovarian samples were taken for histological analysis at intervals from 0 to 5 h post-hCG. GVB was assessed in large antral follicles in complete serial sections of the ovaries. No consistent evidence could be found for an effect of the Npr3 mutation on the kinetics of oocyte maturation in vivo (data not shown). Moreover, ovarian levels of NPPC decreased at the same rate in both Npr3lgj/+ heterozygotes and Npr3lgj/Npr3lgj homozygous mutants; NPPC decreased by 2 h post-hCG (Fig. 10), as previously reported for wild-type mice [12]. When eggs were recovered from oviducts, 14 h post-hCG and fertilized in vitro, the same percentages underwent fertilization and development to the blastocyst stage in vitro: 62/76 (82%) for heterozygotes versus 51/62 (82%) for homozygous mutants. The fertility of Npr3 mutant females was not assessed in consideration of their skeletal abnormalities. Thus, although the longjohn mutation at the Npr3 locus causes significant phenotypic effects on bone growth in homozygotes [19], our studies of this mutation do not provide evidence that NPR3 plays a significant role in the decrease in ovarian NPPC levels or in oocyte development.

FIG. 10.

FIG. 10

Open in a new tab

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.

DISCUSSION

NPPC is a crucial part of the follicular system for maintaining oocytes in meiotic arrest by its interaction with its cognate receptor NPR2 to generate cGMP [7–9], which is transferred to the oocyte where it inhibits PDE3A, thus maintaining levels of cAMP sufficient to sustain meiotic arrest [10, 11]. Furthermore, decreased levels of ovarian NPPC after hCG/LH are correlated with the resumption of meiosis [9, 12]. Thus, the regulation of NPPC levels is integral to coordinating the timing of oocyte maturation with hCG/LH-induced ovulation and the production of an egg competent of undergoing fertilization and embryogenesis. Levels of Nppc mRNA are elevated in vivo in response to stimulation with eCG and then decrease in response to hCG/LH. At the same time that levels of Nppc mRNA and NPPC decrease in vivo in response to hCG/LH, levels of Npr3 mRNA, which encodes an NPPC clearance receptor, increase in MGCs in vivo, suggesting that it may participate in decreasing NPPC levels and regulating oocyte maturation. To further define the factors regulating Nppc and Npr3 mRNA levels in vivo, we assessed the responses of MGCs and cumulus cells to FSH, E2, LH, and ODPFs in vitro.

Because, in rodents, eCG exhibits both FSH and LH activities [24, 25], it was anticipated that one or both of these gonadotropins would stimulate elevated levels of Nppc mRNA in vitro. However, using MGCs isolated from mice not stimulated with eCG and cultured on a substratum of an extracellular matrix (i.e., ECL), neither gonadotropin alone or together promoted Nppc expression by either MGCs or cumulus cells. Rather, E2 alone stimulated expression, an effect that was augmented by FSH in a manner similar to that reported for the control of Npr2 mRNA levels by cumulus cells in vitro [8]. The interactions of FSH and estrogens enhance their actions on granulosa cell development and function (see [32] for review). Estrogen levels increase as a result of eCG treatment in vivo [33, 34]. Moreover, the synthetic estrogen diethylstilbesterol stimulates Nppc and Npr2 expression in rats in vivo [35]. These reports and the findings presented here showing that E2 promotes Nppc expression in vitro, an effect enhanced by FSH, suggest that estrogens produced by gonadotropin stimulation regulate NPPC expression in vivo.

These results implicate E2 as a regulator of the NPPC/NPR2 pathway that is key to the follicular mechanism maintaining meiotic arrest. Nevertheless, to our knowledge, there is no indication from published reports [36–40] (or from our unpublished results) of the follicular/oocyte phenotypes of estrogen receptor 1 (Esr1) or estrogen receptor 2 (Esr2), (also known as estrogen receptor α and β, respectively) or double Esr1/Esr2 deletions or of the Cyp19a1 (aromatase) deletion showing precocious resumption of meiosis in antral follicles. Thus, other pathways could participate, either under the conditions of normal follicular development or in compensation for the absence of ESR1/ESR2 or estrogens, in the mechanisms maintaining meiotic arrest in vivo. Perhaps, in the absence of the classical estrogen receptors, the G protein-coupled receptor GPR30 [41] can mediate the action of estrogens in regulating the actions of NPPC and NPR2 in maintaining meiotic arrest. However, Gpr30 null mice are fully fertile [42].

In cultures of MGCs stimulated with FSH plus E2 to elevate Nppc mRNA expression, EGF, but not LH, caused a decline in both Nppc mRNA and cGMP levels. Nevertheless, production of EGF-like peptides probably occurs within an hour of LH-treatment in vitro. It is possible that the inability of LH to cause this decline in vitro is due to diffusion of LH-induced EGF-like peptides into the culture medium causing a reduction of concentrations to levels unable to act in an autocrine manner. It is unlikely that the lack of response is due to a loss of LH receptors because LH induced the expression of Npr3 mRNA under the same conditions. Stimulation of EGF receptors with EGF caused a rapid decline in Nppc mRNA and cGMP levels, implicating the EGF-receptor pathway in these aspects of the LH response.

There is a similar pattern of Nppc and Lhcgr mRNA expression in ovarian follicles in that both are expressed more highly by MGCs than cumulus cells. ODPFs suppress Lhcgr mRNA levels in cumulus cells and probably account for the higher expression of this transcript by MGCs [17, 29]. We therefore hypothesized that ODPFs are also responsible for the lower expression of Nppc mRNA by cumulus cells. The results presented here failed to support this hypothesis because ODPFs did not suppress Nppc mRNA levels by cumulus cells in vitro but, rather, actually promoted the expression of this transcript. Therefore, mechanisms other than suppression by ODPFs must regulate Nppc expression at levels that are higher in MGCs than in cumulus cells. Instead, regional or cell type-specific factors, or both, must increase Nppc expression by the MGCs to levels higher than in cumulus cells. For example, FSH levels may be higher regionally among the MGCs than the cumulus cells. FSH enters from outside of the follicles and binding to FSH receptors abundant on MGCs could progressively reduce the FSH concentration before reaching the cumulus cells. This would augment the action of estrogens and elevate Nppc mRNA transcripts in MGCs more than in cumulus cells.

At the same time that Nppc expression declines after hCG administration in vivo, expression of Npr3 mRNA, encoding an NPPC clearance receptor, increases. This suggests that NPR3 might be a regulator of ovarian NPPC levels and, therefore, oocyte maturation. In anticipation of this, we assessed the kinetics of hCG-induced meiotic resumption and NPPC decline in Npr3lgj mutant females. Homozygous mutant females exhibit obvious skeletal abnormalities showing that the mutation has clear phenotypic consequences. However, we were unable to detect any significant differences in either oocyte development or ovarian NPPC levels comparing the longjohn Npr3 mutant and heterozygous controls. Hence, decreased ovarian NPPC levels may be a consequence of decreased Nppc mRNA and/or the action of proteases produced downstream of hCG/LH stimulation. Thus, contrary to our hypothesis, and based on the findings presented here, we conclude that NPR3 is not a major regulator of oocyte maturation. Nevertheless, expression of Npr3 is obviously tightly regulated, although its function remains unknown. Perhaps NPR3 acts through some pathway other than one regulating cGMP levels, as reviewed by Rose and Giles [43], to affect processes downstream of hCG/LH stimulation. Oocytes produced in the Npr3lgj mutant females underwent apparently normal fertilization and preimplantation development in vitro. The fertility of these mutants was not assessed because the skeletal and other likely systemic defects would confound the interpretation.

The results presented here, taken together with those of previous studies, form the basis for the following model: gonadotropins via estrogens promote the expression of Nppc and Npr2 by MGCs and cumulus cells and elevate the levels of cGMP in the total granulosa cell compartment (MGCs plus cumulus cells). Cyclic GMP diffuses via gap junctions to the oocyte where it inhibits PDE3A activity, thus sustaining cGMP levels sufficient for maintaining meiotic arrest. This NPPC/NPR2-generated cGMP probably also participates in other processes essential for follicular development [13]. After stimulation by hCG/LH, levels of Nppc mRNA and NPPC decline rapidly. This decline, together with modification of the NPR2 protein such that its activity is decreased [12], results in decreased production of cGMP, constituting an important part of a complex network of processes participating in triggering oocyte meiotic maturation. Although the expression of Npr3, encoding an NPPC clearance receptor, dramatically increases after hCG/LH stimulation, it does not appear to be a major player in this network.

ACKNOWLEDGMENT

We thank Drs. Mary Ann Handel and You-Qiang Su for their helpful suggestions and Marilyn O'Brien for her expert assistance.

Footnotes

1Supported by grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Grants HD23839 and HD21970 (K.B.L., K.S., K.W., and J.J.E.) and HD014939 (T.U. and L.A.J.) and the National Basic Research Program of China 2012CB944401 (M.Z.).

REFERENCES

  1. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 2004; 303: 682 684 [DOI] [PubMed] [Google Scholar]
  2. Conti M, Hsieh M, Park JY, Su YQ. Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 2006; 20: 715 723 [DOI] [PubMed] [Google Scholar]
  3. Shimada M, Hernandez-Gonzalez I, Gonzalez-Robayna I, Richards JS. Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol Endocrinol 2006; 20: 1352 1365 [DOI] [PubMed] [Google Scholar]
  4. Hsieh M, Lee D, Panigone S, Horner K, Chen R, Theologis A, Lee DC, Threadgill DW, Conti M. Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation. Mol Cell Biol 2007; 27: 1914 1924 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Panigone S, Hsieh M, Fu M, Persani L, Conti M. Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway. Mol Endocrinol 2008; 22: 924 936 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hsieh M, Thao K, Conti M. Genetic dissection of epidermal growth factor receptor signaling during luteinizing hormone-induced oocyte maturation. PLoS One 2011; 6: e21574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Zhang M, Su YQ, Sugiura K, Xia G, Eppig JJ. Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science 2010; 330: 366 369 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Zhang M, Su YQ, Sugiura K, Wigglesworth K, Xia G, Eppig JJ. Estradiol promotes and maintains cumulus cell expression of natriuretic peptide receptor 2 (NPR2) and meiotic arrest in mouse oocytes in vitro. Endocrinology 2011; 152: 4377 4385 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kawamura K, Cheng Y, Kawamura N, Takae S, Okada A, Kawagoe Y, Mulders S, Terada Y, Hsueh AJ. Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes. Hum Reprod 2011; 26: 3094 3101 [DOI] [PubMed] [Google Scholar]
  10. Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, Wang H, Ke H, Nikolaev VO, Jaffe LA. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 2009; 136: 1869 1878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Vaccari S, Weeks JL, II,, Hsieh M, Menniti FS, Conti M. Cyclic GMP signaling is involved in the luteinizing hormone-dependent meiotic maturation of mouse oocytes. Biol. Reprod 2009; 81: 595 604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Robinson JW, Zhang M, Shuhaibar LC, Norris RP, Geerts A, Wunder F, Eppig JJ, Potter LR, Jaffe LA. Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes. Dev Biol 2012; 366: 308 316 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sato Y, Cheng Y, Kawamura K, Takae S, Hsueh AJ. C-Type natriuretic peptide stimulates ovarian follicle development. Mol Endocrinol 2012; 26: 1158 1166 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geister KA, Brinkmeier ML, Hsieh M, Faust SM, Karolyi IJ, Perosky JE, Kozloff KM, Conti M, Camper SA. A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type. Hum Mol Genet 2013; 22: 345 357 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kiyosu C, Tsuji T, Yamada K, Kajita S, Kunieda T. NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary. Reproduction 2012; 144: 187 193 [DOI] [PubMed] [Google Scholar]
  16. Tsuji T, Kiyosu C, Akiyama K, Kunieda T. CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR-mediated signaling in preovulatory follicles. Mol Reprod Dev 2012; 79: 795 802 [DOI] [PubMed] [Google Scholar]
  17. Diaz FJ, Wigglesworth K, Eppig JJ. Oocytes determine cumulus cell lineage in mouse ovarian follicles. J Cell Sci 2007; 120: 1330 1340 [DOI] [PubMed] [Google Scholar]
  18. Potter LR. Natriuretic peptide metabolism, clearance and degradation. FEBS J 2011; 278: 1808 1817 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jaubert J, Jaubert F, Martin N, Washburn LL, Lee BK, Eicher EM, Guenet JL. Three new allelic mouse mutations that cause skeletal overgrowth involve the natriuretic peptide receptor C gene (Npr3). Proc Natl Acad Sci U S A 1999; 96: 10278 10283 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 1990; 138: 16 25 [DOI] [PubMed] [Google Scholar]
  21. Sugiura K, Su YQ, Eppig JJ. Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression. Mol Reprod Dev 2009; 76: 537 547 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Su YQ, Sugiura K, Woo Y, Wigglesworth K, Kamdar S, Affourtit J, Eppig JJ. Selective degradation of transcripts during meiotic maturation of mouse oocytes. Dev Biol 2007; 302: 104 117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Eppig JJ, Wigglesworth K, Pendola FL. The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci U S A 2002; 99: 2890 2894 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Licht P, Gallo AB, Aggarwal BB, Farmer SW, Castelino JB, Papkoff H. Biological and binding activities of equine pituitary gonadotrophins and pregnant mare serum gonadotrophin. J Endocrinol 1979; 83: 311 322 [DOI] [PubMed] [Google Scholar]
  25. Racowsky C. Gamete resources: origin and production of oocytes. : Pedersen RA, McLaren A, First NL. (eds.), Animal Applications of Research in Mammalian Development. Current Communications in Cell and Molecular Biology, vol. 4. Plainview, NY: Cold Spring Harbor Laboratory Press; 1991: 23 82 [Google Scholar]
  26. Parrott JA, Vigne JL, Chu BZ, Skinner MK. Mesenchymal-epithelial interactions in the ovarian follicle involve keratinocyte and hepatocyte growth factor production by thecal cells and their action on granulosa cells. Endocrinology 1994; 135: 569 575 [DOI] [PubMed] [Google Scholar]
  27. Eppig JJ, Chesnel F, Hirao Y, O'Brien MJ, Pendola FL, Watanabe S, Wigglesworth K. Oocyte control of granulosa cell development: how and why. Hum Reprod 1997; 12: 127 132 [PubMed] [Google Scholar]
  28. Li R, Norman RJ, Armstrong DT, Gilchrist RB. Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells. Biol Reprod 2000; 63: 839 845 [DOI] [PubMed] [Google Scholar]
  29. Eppig JJ, Wigglesworth K, Pendola FL, Hirao Y. Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells. Biol Reprod 1997; 56: 976 984 [DOI] [PubMed] [Google Scholar]
  30. Norris RP, Freudzon M, Nikolaev VO, Jaffe LA. Epidermal growth factor receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to LH. Reproduction 2010; 140: 655 662 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Davis JS, Weakland LL, West LA, Farese RV. Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone. Biochem J 1986; 238: 597 604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Richards JS. Perspective: the ovarian follicle—a perspective in 2001. Endocrinology 2001; 142: 2184 2193 [DOI] [PubMed] [Google Scholar]
  33. Davis SR, Burger HG, Robertson DM, Farnworth PG, Carson RS, Krozowski Z. Pregnant mare's serum gonadotropin stimulates inhibin subunit gene expression in the immature rat ovary: dose response characteristics and relationships to serum gonadotropins, inhibin, and ovarian steroid content. Endocrinology 1988; 123: 2399 2407 [DOI] [PubMed] [Google Scholar]
  34. Leveille MC, Armstrong DT. Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions. Gamete Res 1989; 23: 127 138 [DOI] [PubMed] [Google Scholar]
  35. Noubani A, Farookhi R, Gutkowska J. B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells. Endocrinology 2000; 141: 551 559 [DOI] [PubMed] [Google Scholar]
  36. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O. Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci U S A 1998; 95: 15677 15682 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar M, Mayo KE, Korach KS. Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult. Endocrinology 1999; 140: 2733 2744 [DOI] [PubMed] [Google Scholar]
  38. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. Development 2000; 127: 4277 4291 [DOI] [PubMed] [Google Scholar]
  39. Britt KL, Drummond AE, Dyson M, Wreford NG, Jones MEE, Simpson ER, Findlay JK. The ovarian phenotype of the aromatase knockout (ArKO) mouse. J Steroid Biochem Mol Biol 2001; 79: 181 185 [DOI] [PubMed] [Google Scholar]
  40. Huynh K, Jones G, Thouas G, Britt KL, Simpson ER, Jones ME. Estrogen is not directly required for oocyte developmental competence. Biol Reprod 2004; 70: 1263 1269 [DOI] [PubMed] [Google Scholar]
  41. Wang C, Prossnitz ER, Roy SK. Expression of G protein-coupled receptor 30 in the hamster ovary: differential regulation by gonadotropins and steroid hormones. Endocrinology 2007; 148: 4853 4864 [DOI] [PubMed] [Google Scholar]
  42. Otto C, Fuchs I, Kauselmann G, Kern H, Zevnik B, Andreasen P, Schwarz G, Altmann H, Klewer M, Schoor M, Vonk R, Fritzemeier KH. GPR30 does not mediate estrogenic responses in reproductive organs in mice. Biol Reprod 2009; 80: 34 41 [DOI] [PubMed] [Google Scholar]
  43. Rose RA, Giles WR. Natriuretic peptide C receptor signalling in the heart and vasculature. J Physiol 2008; 586: 353 366 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biology of Reproduction are provided here courtesy of Oxford University Press

RESOURCES