| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Laboratory of Cardiovascular Biochemistry and
b Laboratory of Cellular Biology of Hypertension, Centre Hospitalier de l'Université de Montréal, Campus Hôtel-Dieu, Department of Medicine, University of Montreal, Quebec, Canada H2W 1T8
c Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1T8
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ß, activin (24-kDa protein with structural homology to transforming growth factor ß), angiotensin II, vasoactive intestinal peptide, inhibin, substance P, endothelin, and others [2]. To this vast array of regulatory factors, the natriuretic peptide (NP) system has been recently added [3–8]. Atrial natriuretic peptide (ANP), originally isolated from mammalian atrial cardiocytes, has been shown to elicit diuretic, natriuretic, and vasorelaxant effects [9]. Although ANP was the first natriuretic factor to be identified, two additional peptides showing structural homology and pharmacological properties similar to those of ANP were isolated from porcine brain. Thus, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and ANP constitute the NP family [10, 11]. Cloning of complementary DNA indicates that the three members of the NP family are derived from different genes. The physiological activities of NPs are mediated by two specific plasma membrane receptors, guanylyl cyclase-type receptors GC-A and GC-B [12]. The molecular structures of GC-A and GC-B receptors are similar [13]. Their cytosolic region contains a guanylyl cyclase and a kinase-like domain. The extracellular parts of these receptors exhibit higher affinity either to ANP (GC-A) or to CNP (GC-B) [14]. The activation of the receptors by NPs results in cGMP production [15]. A third "clearance" receptor has also been cloned [16]. This receptor lacking intracellular cyclase and kinase domains has no effect on cGMP production and is believed to clear NPs from plasma [16]. The clearance receptor does not discriminate, binding all NPs with similar affinity. However, evidence exists that this receptor may have other bioactivities because it inhibits adenylyl cyclase activity and cAMP production [17].
While the initial demonstration of properties of ANP focused on its role in fluid and salt homeostasis and cardiovascular functions, it appeared that the profile of effects that ANP exerts is much more diverse. ANP has been shown to have hypothalamic action to alter LH release [18]. In the gonads, ANP has been reported to stimulate progesterone secretion from human granulosa luteal cells [3]. Furthermore, ANP has been shown to have potent growth-regulatory properties [19].
Several lines of evidence suggest some important role for this peptide in ovarian function in mammals, including humans. All known components of the ANP system have been identified in ovaries [6]. Furthermore, we have shown changes in the NP system during the ovarian cycle [8]. Immunoreactive ANP is present in interstitial-theca cells of the rat ovary [5] and pig ovarian granulosa cells [7]. The presence of immunoreactive ANP was demonstrated in follicular fluid of pig ovarian follicles and rabbit ovarian homogenates and perfusates [5], and its biologically active receptors have been characterized in adult ovaries [6]. Cultured granulosa cells respond to physiological levels of ANP by increasing intracellular cGMP formation and progesterone secretion [3, 4]. However, the role of NPs in reproductive functions is not yet known. The present study was designed to gain further insight into the physiological role of NP in ovarian functions. To this end we investigated the alterations of the NP system during follicular growth and ovulation induced by gonadotropin stimulation. As such a regimen is also used in clinical treatment, we believe that our data may be of relevance to fertility/infertility.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immature female Sprague-Dawley rats were injected s.c. with eCG (20 IU in 0.2 ml) on the 23rd day after birth. Ovulation was induced 48 h later by s.c. injection of 20 IU of hCG [20]. Animals were killed by decapitation after treatment with eCG alone (48 h later) or with eCG+hCG (5 days later). Rats injected with the same volume of saline (0.9% solution) served as control. The ovaries were quickly dissected, cleaned, weighed, and frozen in liquid nitrogen and stored at -80°C for membrane preparation, and for mRNA extraction. In a separate experiment, animals were treated with eCG (20 IU) and decapitated 1, 2, 6, 12, and 48 h following treatment, and their ovaries were dissected and frozen in liquid N2 for mRNA extraction, or in ice-cold isopentane for autoradiographic studies.
The effects of gonadotropin treatment on ovarian morphology is well known [2] and was verified by light microscopy performed on hematoxylin- and eosin-stained ovarian sections.
Preparation of Ovarian Membranes
The ovarian membranes were prepared for binding as previously described [6]. Frozen ovaries were homogenized twice for 10 sec in ice-cold Tris-HCl buffer (25 mM, pH 7.4, 30 mM sucrose, 1 mM MnCl2, and 0.5 mM PMSF) with a Polytron PT-10 (Brinkmann Instruments, Rexdale, ON, Canada) and centrifuged at 2500 x g. The supernatant was then centrifuged for 30 min at 30 000 x g at 4°C. The pellet was washed and aliquots were used for protein determination.
Membrane Binding Studies
Ovarian membrane suspensions were prepared at a concentration of 75 µg protein/100 µl in Tris-HCl buffer (50 mM, pH 7.4, 0.1% MgCl2, 0.49% MnCl2, 0.04% Na2 EDTA, 0.4% BSA, 0.5 mM PSMF, and 0.1% bacitracin). The peptides were iodinated by the lactoperoxidase method and purified by HPLC as previously described [21]. All binding assays were performed in plastic tubes. The membrane suspensions (75 µg protein) were incubated for 90 min with 125I-ANP (18 000–20 000 cpm) in the absence and presence of increasing concentrations (10-12 to 10-6 M) of ANP or synthetic C-ANF, a 5-amino acid ring deleted ANP (des[Gln116, Ser117, Gly118, Leu119, Gly120]ANP102–121) that specifically binds to clearance receptors but not to guanylyl cyclase-type receptors. Binding in the presence of 10-6 M ANP was considered nonspecific. The reaction was stopped by the addition of 2 ml ice-cold Tris-HCl buffer and rapid filtration on Whatman (Clifton, NJ) GF/C filters presoaked in 1% polyethylenimine (Sigma Chemical Co., St. Louis, MO). The filters were then washed three times with 2 ml ice-cold Tris-HCl buffer (50 mM, pH 7.4). Receptor-bound radioactivity was counted in an LKB (Rockville, MD) 
-counter.
Preparation of Ovarian Cell Suspension and cGMP Production
Stimulation of cGMP production by the NPs was evaluated in ovarian cells obtained from control and hormone (eCG or eCG+hCG)-treated rats. Ten female rats in each group were killed by decapitation and their ovaries quickly dissected. The tissues were placed in Dulbecco's modified medium (DME), minced with a scalpel, and dispersed enzymatically in 5 ml DME containing 10 mg collagenase (type 1A; Sigma) and 50 µg DNase (from bovine pancreas; Boehringer Mannheim, Laval, PQ, Canada). The cell suspensions were incubated for 3 h at 37°C in 95% O2:5% CO2; then the cells were dispersed mechanically through a glass pipette, filtered twice through 250-µm and 100-µm nylon filters, and centrifuged at 90 x g for 15 min at 4°C. The pellets were resuspended in fresh medium and centrifuged twice. Cell viability was ascertained by the trypan blue exclusion method, and the cells were counted with a hemacytometer. The dispersed cells were resuspended in DME, and 500 µl of the cell suspension (250 000 cells) was incubated with increasing concentrations (10-11 to 10-6 M) of rat and human ANP (Ser99-Tyr126), C-ANF, porcine and rat BNP, and porcine CNP (all reagents from Peninsula Laboratories, Belmont, CA). The incubations were carried out, in duplicate, for 3 h at 37°C in the presence of 500 µM methylisobutylxanthine with gentle shaking. The reaction was stopped by placing the cells in an ice bath, followed by centrifugation at 250 x g for 5 min at 4°C. Cyclic GMP was measured in the cell medium by RIA as described previously [15] using three different dilutions.
Autoradiography
Whole frozen ovarian sections (20 µm) were cut and mounted on acid-washed gelatinized slides and then placed overnight in a partial vacuum at -4°C. Slides were stored in boxes with Drierite (Aldrich Chemical Co., Milwaukee, WI) at -80°C until the autoradiographic procedures were performed [22]. Optimal binding conditions (amount of radiolabeled ligand and incubation time) were determined in preliminary studies. Sections from control and eCG-treated rat ovaries were assayed simultaneously. Duplicate slides were brought to room temperature in incubation buffer containing 50 mM Tris-HCl, pH 7.4, and 0.1% polyethylenimine for 15 min. The slides were incubated with 50 pM 125I-ANP for 1 h at room temperature. The binding buffer consisted of 50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 40 µg/ml bacitracin, and 0.5% BSA. The sections were washed four times for 2 min each with ice-cold Tris-HCl buffer, pH 7.4, at 4°C; they were then rinsed with distilled water to remove the salts and dried rapidly under a stream of cold air. Nonspecific binding was determined on adjacent sections under identical incubation conditions, except for the addition of 10-6 M ANP. Localization of the CNP-binding sites was also determined in sections incubated with 125I-tyroCNP (100 pM) and inhibition of binding with 10-6 M CNP.
After exposure to a phosphor-sensitive cassette, the dried tissue sections were dipped in Kodak (Eastman Kodak, Rochester, NY) NTB-2 emulsion (diluted 1:1 with distilled water). Emulsion-coated slides were allowed to dry in the dark and were stored in light-tight boxes at -80°C for 1 wk. The slides were developed and stained with hematoxylin. Slides were examined, by brightfield microscopy, for the localization of silver grains, and representative structures were photographed.
Quantitative Polymerase Chain Reaction Measurement of GC-A and GC-B mRNA Levels
Ovarian tissues RNA was isolated according to the guanidinium thiocyanate-phenol-chloroform method, as described elsewhere [23]. The concentration of each RNA sample was determined by UV absorbance at 260 nm. GC-A and GC-B mRNA levels were determined by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method described previously [24]. In brief, total RNA preparations were mixed with fixed amounts of a synthetic mutated RNA that contains an EcoRI site. The RT reaction product was subjected to PCR amplification with the following primers: GC-A forward, 5'-AAGCTTATCTGGAGGAGAAGCGCA-3'; GC-A reverse, 5'-TCAGCCTCGAGTGCTACATCCCCG-3'; GC-B forward, 5'-GGTACCAGCATATTGGACAACCTC-3'; GC-B reverse, 5'-CAGGAGTCCAGGAGGTCCTTTTCG-3'.
A reaction volume of 50 µl containing 50 mM Tris-HCl buffer (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 40 pmol of each primer, 2.5 U of Taq DNA polymerase (Gibco, BRL, Oakville, ON, Canada), and 10 µCi [32P]dCTP was incubated for 30 cycles of 1-min denaturation at 94°C, 1-min annealing at 65°C, and 1.5-min polymerization at 72°C; 10 µl of each sample was digested at 37°C for 1 h with 10 U of EcoRI. The digested samples were electrophoresed on 1.5% agarose gel, giving an upper nondigested band derived from endogenous mRNA (GC-A, 677 base pairs [bp]; GC-B, 762 bp). Lower digested bands were derived from mutated cRNA (GC-A, 373 and 304 bp; GC-B, 385 and 377 bp, respectively). Corresponding radioactive bands were counted with ImageQuant (Molecular Dynamics, Sunnyvale, CA) software. Relative mRNA products were represented as a percentage of values obtained from cRNA amplification.
In preliminary experiments, RNA samples were amplified in aliquots containing increasing concentrations of mutated cRNA. The determined 50% titration point, the point at which mRNA and cRNA curves intersect, indicated the number of mRNA molecules in the sample (3 x 105 molecules of GC-A and 5 x 107 molecules of GC-B). This number of cRNA molecules was selected arbitrarily as an internal standard for semiquantitative comparison of GC-A and GC-B mRNA levels in ovarian samples.
PCR Amplification of ANP and CNP Transcripts
PCR reactions were conducted according to previously described procedures [25]. Briefly, total RNA was digested by RQ1 DNase (Promega, Madison, WI) and subjected to RT. Then, the normalized amounts of cDNA from tested samples were amplified with liver genomic DNA and 700 ng each of forward and reverse primers (corresponding to sequences in the first and second exons of studied genes) and with 2.5 U of Taq DNA polymerase (Perkin-Elmer-Cetus, Norwalk, CT). The following primers were used for PCR amplification reaction: ANP forward, 5'-CAGCATGGGCTCCTTCTCCA-3'; ANP reverse, 5'-GTCAATCCTACCCCCGAAGCAGCT-3'; CNP forward, 5'-CGCACCATGCACCTCTCCCAGCTGAT-3'; CNP reverse, 5'-CGCTGCACTAACATCCCAGACCGC-3'.
The volume and buffer composition was the same as described for PCR amplification of GC-A and GC-B transcripts. Reaction mixture with final concentration of 0.2 mM dNTPs and 10 µCi 32P was amplified at 25–30 cycles of 1 min at 94°C, 1 min at 63°C, and 3 min at 72°C. PCR reaction product electrophoresed on 1.5% agarose gave two bands: the upper band was derived from genomic DNA (ANP: 534 bp, CNP: 824 bp), and the lower band was derived from amplified cDNA (ANP: 430 bp, CNP: 403 bp).
Effect of ANP on Estradiol and Progesterone Release
Ovaries from control and eCG-treated rats were cut in four pieces, washed, and incubated in 1 ml of minimal essential medium. Tissue incubation was performed at 37°C for 3 h in a humidified atmosphere containing 5% CO2:95% O2. The incubation was stopped by rapid cooling on ice. Estradiol (E2) and progesterone were measured in the medium by RIA using commercially available kits.
Data Analysis
The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) for the ligands used in the competitive binding radioreceptor studies were calculated by the nonlinear method using the LIGAND computer program (Elsevier-Biosoft, Cambridge, UK). Data storage, graphical output, and statistical analysis assessed by one-way ANOVA were accomplished using RS1 data analysis software (BBN, Cambridge, MA). Statistical significance was taken as p < 0.05. All data are reported as mean ± SEM.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
NP Receptors
Hormonal treatment did not result in qualitative changes in NP receptor subtypes. The ovarian membranes, before and after hormone treatment, showed the presence of NP guanylyl cyclase-type receptors exclusively. C-ANF, a specific ligand for the clearance receptor, did not compete with 125I-ANP, which is consistent with the absence of clearance receptors. Figure 1 shows that the binding of 125I-ANP to ovarian membranes was increased significantly (p < 0.05) after both hormonal treatments, with B/T (bound/total amount of radioactivity used) representing 14.0 ± 2.0% and 13.4 ± 1.0% as compared to 8.2 ± 1.5% in control rats, whereas affinity was not affected by the treatments. The increased total binding was mainly influenced by increased receptor number. Calculation of the kinetic parameters of the competitive binding curves (Table 1) revealed that maximum binding capacity (Bmax) was 225 ± 50 fmol/mg protein in control rats, and that after hormonal treatment it increased to 354 ± 51 and 492 ± 130 fmol/mg protein (p < 0.05) in eCG and eCG+hCG treatment, respectively, with no changes in the dissociation constant.
|
|
Stimulation of cGMP Production by NPs in Dispersed Ovarian Cells
Production of cGMP in ovarian cells was determined in control and gonadotropin-treated rats. Basal levels of cGMP production in ovarian cells of eCG-treated rats were increased to 5-fold (0.16 ± 0.04 to 0.87 ± 0.08 pmol/ml) in relation to the level in corresponding untreated control rats (25-day-old rats). However, after ovulation and corpus luteum formation by eCG+hCG treatment, the production of cGMP was decreased to 0.18 ± 0.08 pmol/ml from 0.92 ± 0.21 pmol/ml in corresponding age-matched control rats. The difference in basal cGMP production between 25- and 30-day-old control animals is most likely due to endogenous developmental hormonal changes.
Since the hormonal treatments resulted in different basal levels of cGMP production, the results are presented in fold increase over basal. Figure 2 shows that the ability of NPs to activate guanylyl cyclase-type receptors in dispersed rat ovarian cells was significantly increased in both eCG (Fig. 2A)- and eCG+hCG (Fig. 2B)-treated as compared to respective control ovaries. In eCG-treated rats, both ANP and CNP stimulated ovarian cGMP production to more than 3-fold above basal levels. Stimulation of cGMP production by ANP started at 10-9 M (1.63 ± 0.06 pmol/ml) and was maximal at 10-8 M concentration of peptide (3.15 ± 0.06 pmol/ml, p < 0.0001 vs. basal); and CNP at 10-8 M and 10-6 M progressively increased cGMP production to 1.52 ± 0.01 and 2.82 ± 0.12 pmol/ml (p < 0.0001), respectively. On the other hand, stimulation by 10-8 M ANP increased cGMP production to 3.46 ± 0.31 pmol/ml or 18-fold over basal levels (p < 0.001) in ovarian cells from eCG+hCG-treated rats, whereas production of cGMP by CNP was observed only at 10-6 M (Fig. 2B). The stimulation of cGMP by BNP paralleled that observed with ANP in all groups, confirming that BNP and ANP bind to the same receptor type. C-ANF had no effect on cGMP production in both hormone-treated and control ovarian cells (not shown).
|
Autoradiography
The distribution of receptors for the NPs was examined in emulsion-dipped and developed slides. Silver grains, corresponding to both 125I-tyr oCNP and 125I-ANP binding, were present in small follicles in control ovaries (Fig. 3). Figure 3 shows binding of 125I-ANP and 125I-tyr oCNP observed in preantral follicles of control ovaries. Treatment with eCG resulted in significantly increased binding of the granulosa cell layer of antral follicles. However, higher binding was observed with 125I-tyroCNP.
|
Quantitation of GC-A and GC-B mRNA by RT-PCR
Quantitative measurement of GC-A and GC-B mRNA molecules was performed in ovaries from control, eCG-, and eCG+hCG-treated rats. Figure 4 includes a representative PhosphorImager (Molecular Dynamics) scan of PCR products after EcoRI digestion and agarose gel electrophoresis. The upper bands represent endogenous nondigestable cDNA, and lower digested bands represent mutated cDNA. As shown in Figure 4A, 48-h eCG treatment decreased GC-A by 50% (from 8 x 106 to 4 x 106 molecules/µg of total mRNA) and had no effect on GC-B mRNA, but eCG+hCG treatment increased ovarian GC-A mRNA from 2.3 x 106 to 3.5 x 106 molecules/µg total mRNA compared to the control value. However, the number of ovarian GC-B mRNA molecules decreased from 1.7 x 108 in control to 5.8 x 107 molecules/µg RNA of eCG+hCG-treated rats (Fig. 4).
|
These findings show that mRNA of ovarian NP receptors are not changed or may be even decreased by the treatments. Considering, in contrast, the significant increase in the number of NP receptors observed in membrane binding studies after eCG treatment, it was hypothesized that the changes observed at the transcriptional level may appear earlier in time. Therefore, further experiments were performed to examine the time-dependent changes in ovarian mRNA for both GC-A and GC-B. Rat ovarian total RNA from control and eCG-treated rats were extracted at 0, 1, 2, 6, 12, 24, and 48 h after treatment. To measure GC-A and GC-B semiquantitatively, 5 x 106 GC-A cRNA or 1 x 108 GC-B cRNA mutated molecules (representing the 50% titration point) were added to 1 µg of total ovarian RNA before RT and PCR. As shown in Figure 5, both GC-A and GC-B mRNA levels started to increase at 2 h and reached maximal levels at 6 h after eCG treatment. Increased levels of mRNA for GC-B but not for GC-A mRNA were still observed 12 h after eCG injection, and at 24 and 48 h the receptor mRNA levels were below basal.
|
Quantitation of ANP and CNP mRNA by RT-PCR
We investigated whether the stimulation of ovarian GC-A and GC-B mRNA during time–course eCG treatment was paralleled by activation of ovarian ANP and CNP gene expression. For this purpose ANP mRNA and CNP mRNA were measured semiquantitatively by competitive RT-PCR. Rat liver genomic DNA (gDNA, 120 ng) served as an internal standard for the inhibition of 20 ng of cDNA prepared from ovarian total RNA. The PCR products electrophoresed on 1.5% agarose gave 2 bands: an upper band derived from gDNA (ANP: 534 bp, CNP: 824 bp), and a lower band derived from amplified cDNA (ANP: 430 bp, CNP: 403 bp). The radioactive bands were quantified with a PhosphorImager. The ratio between counts given by the target and competitor products was calculated, and PCR product ratios (cDNA/gDNA) were presented as ANP or CNP mRNA arbitrary units. The arbitrary units were plotted versus time after treatment with eCG. Results were normalized to time zero taken as 1.0. Figure 6 shows that eCG treatment resulted in an increased ovarian expression of both ANP and CNP genes. A slight increase in ANP mRNA was observed as early as 1 h after eCG injection and persisted up to 6 h. The increase in CNP mRNA was observed at 2 h after eCG treatment and peaked (5-fold increase) at 6 h after injection. Increased CNP mRNA was observed up to 48 h, when maximal follicular maturation and antrum formation are known to occur and set the stage for impending ovulation induction by LH/hCG action.
|
Effect of ANP on E2 and Progesterone Production
To assess the possibility that ANP may affect hormonal (E2 and progesterone) production, the ovaries were incubated with 10-6 M ANP. Figure 7 shows that accumulation of E2 in the medium was significantly higher in the ovaries from eCG-treated animals than in those from the control group. However, while it had no effect on the control and eCG+hCG groups, ANP significantly inhibited E2 accumulation in the ovaries of eCG-treated animals. On the other hand, progesterone accumulation in the medium increased by the treatments was not altered by ANP in control (0.62 ± 0.10 vs. 0.81 ± 0.23 ng/ml), eCG-treated (12.4 ± 2.7 vs. 11.8 ± 2.6 ng/ml), or eCG+hCG-treated rats (72.2 ± 16.6 vs. 82.6 ± 25.3 ng/ml).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present data demonstrate that eCG treatment promptly activated the ovarian NP system in a number of ways: 1) the number of guanylyl cyclase-type receptors (which are the exclusive NP receptors in the rat ovary) were increased, as determined by binding studies and by cGMP accumulation through NP stimulation; 2) the number of both GC-A and GC-B receptor subtypes localized on granulosa cells increased; 3) the expression of GC-B receptors was more abundant than that of GC-A receptors in both control and gonadotropin-stimulated rats; 4) peak levels of mRNA for both GC-B and GC-A receptors were reached 6–12 h following eCG treatment; 5) receptor up-regulation was associated with a 5-fold increase of CNP mRNA and a 2-fold increase in ANP mRNA; 6) ANP had a modulatory effect on eCG-induced ovarian steroidogenesis by inhibiting E2 production.
Our present and previously published data [8] show a coordinated hormonal stimulation of the NPs and their receptors in the rat ovary. CNP in the ovary is most likely due to local synthesis since CNP is not detected or circulates in very low concentrations. Together with the presence of guanylyl cyclase B receptor subtypes in the ovary and the responsiveness to hormonal regulation, CNP and GC-B receptors emerge as specific regulators of ovarian functions. In this context, it is important to note that the gene expression of both GC-B receptor [27] and its natural ligand, CNP [28], is higher in ovaries than in other tissues in the rat.
We have not found in the present experiments a good parallelism between receptor mRNA level on one hand and binding of ovarian NP receptors and cGMP stimulation on the other. This is not an isolated observation, as all receptors may not be functionally coupled to the transducers. The apparent lack of correlation has been already noted in other tissues, including the uterus [29]. However, the heterogeneity of NP receptors may be responsible for this phenomenon. It has been observed that in tissues in which the C receptor is absent, lower concentrations of ANP appear to be needed to increase cGMP levels. GC-A and GC-B expression in COS-7 cells demonstrates the same difference between the EC50 of cGMP stimulation (~100 nM) and the Kd of peptide binding (0.01 µM) [30]. Thus, this appears to be a property of this family of peptide receptors. Different mechanisms, such as oligomerization of receptors, intramolecular inhibitory domain, and existence of essential cofactor have been proposed to explain this phenomenon. Another possibility is that receptor mRNAs were activated at 6 or 12 h following treatment, prior to the appearance of the active protein later at 48 h.
The role of the NP system in the ovary is not fully understood. However, NPs bind to their receptors and activate guanylyl cyclase (as determined by cGMP accumulation) and inhibit cAMP production. In the present study, cGMP accumulation was increased by eCG treatment but significantly decreased by the addition of hCG, consistent with previous reports [31]. Previous studies [32, 33] have already shown a correlation between the changes in ovarian cGMP and steroid hormones during the hamster estrus cycle. The highest cGMP level, measured in the largest follicles, was observed at 0900 h at the day of proestrus when E2 was already rising, reaching its peak at 1400 h. This observation is in agreement with our previous report showing that the level of CNP during proestrus was about 4-fold higher than the levels measured at any of the stages of the cycle. Similarly, GC-A and GC-B mRNAs were highest in the ovaries at proestrus, being 2- to 3-fold higher than the level seen at estrus. These observations indicate that there is a temporal interrelation between the increase in ovarian E2, NPs, and cGMP. Therefore, it is conceivable that the activation of the ovarian NP system is involved in ovarian function through cGMP increase during proestrus or the exaggerated proestrus mimicked by eCG treatment.
A functional role of cGMP in inhibition of spontaneous oocyte maturation has been postulated [34]. Subsequently, Tornell et al. [35] demonstrated that ANP-stimulated accumulation of cGMP in isolated rat oocyte-cumulus cellular complexes led to inhibition of spontaneous oocyte maturation. Such a preemptive action may serve to prevent untimely maturation until all other follicular components including pressure are ready for ovulation. This mechanism leading to inhibition of some secretory components could explain in part the decreased E2 secretion attributed to ANP in eCG-stimulated ovaries. On the other hand, cGMP may act directly via inhibition of cAMP-induced aromatase activity as has been shown by LaPolt and Hong [36]. In this regard, the inhibitory effect of atrial NP merits consideration, since adrenal steroidogenesis has been associated with an increase in cGMP-stimulated phosphodiesterase activity and decline in cAMP [36].
Recently cGMP has been shown to modulate the macromolecule permeability by decreasing albumin flux across the monolayer of the microvascular coronary endothelial cells [37]. Further studies showed that this effect is calcium dependent [38]. In basal conditions, cGMP decreases endothelial permeability, while in the presence of elevated Ca2+, cGMP causes an increase in permeability in aortic endothelial cells [38]. It is interesting to note that CNP has been found in, and could be released from, endothelial cells [39]. Therefore it is conceivable that ovarian CNP could be involved in the alteration of cellular permeability that leads to antrum formation, which is essentially a buildup of fluid along with accumulation of albumin in the follicles that are destined for ovulation.
Recent work has demonstrated that activation of the cGMP pathway in cultured preantral follicles prevents programmed cell death [40]. It was also suggested that the anti-apoptotic cGMP pathway is regulated by multiple mechanisms at different stages of follicular development, including roles for as-yet-unidentified factors. Therefore, it could be speculated that CNP and ANP acting as the physiological stimulators of guanylyl cyclase may prevent apoptosis in addition to regulating other functions in ovarian follicles. In fact, treatment of cultured follicles with FSH in combination with a cGMP analogue resulted in a significant increase in the number of viable cells and in cell differentiation [39]. Findings such as high ANP in human follicular fluid that harbored fertilizable oocytes may also suggest protective action as discussed above, in addition to other possibilities [41].
The results reported here were obtained in cell suspension of whole ovaries, where the cellular interactions may influence the response to ligands. But it will be of equal interest in a subsequent study to analyze NP receptor levels in purified granulosa and theca-interstitial cells. Indeed, it will be of interest to examine the NP system among different subpopulations of granulosa cells in a given follicle, as these cells are known to have different functional properties and arise from follicles that have been programmed to support oocyte development in dominant follicles preselected to undergo ovulation.
In summary, the present study shows that the ovarian NP system is under gonadotropin regulation particularly by FSH, a crucial hormone necessary for folliculogenesis. Furthermore, CNP and GC-B receptors emerge as an important intraovarian regulatory system.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Jolanta Gutkowska, Laboratory of Cardiovascular Biochemistry, CHUM Research Center, Campus Hotel-Dieu, 3850 St-Urbain Street, Pavillon Masson, Montreal, PQ, Canada H2W 1T8. FAX: 514 843 2715; gutkowsj{at}ere.umontreal.ca
Accepted: February 22, 1999.
Received: August 14, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M Zhang, Y Tao, B Zhou, H Xie, F Wang, L Lei, L Huo, Q Sun, and G Xia Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways J. Mol. Endocrinol., April 1, 2005; 34(2): 459 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Jankowski, D Wang, S Mukaddam-Daher, and J Gutkowska Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle J. Endocrinol., January 1, 2005; 184(1): 209 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. R. Costa, C. R. Fagundes-Moura, V. M. Pereira, L. F. Silva, M. A. R. Vieira, R. A. S. Santos, and A. M. Dos Reis Angiotensin-(1-7): A Novel Peptide in the Ovary Endocrinology, May 1, 2003; 144(5): 1942 - 1948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chen and D. G. Gardner Osmoregulation of Natriuretic Peptide Receptor Signaling in Inner Medullary Collecting Duct. A REQUIREMENT FOR p38 MAPK J. Biol. Chem., February 15, 2002; 277(8): 6037 - 6043. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
