B-Type Natriuretic Peptide Receptor Expression and Activity Are ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 2 Printed in U.S.A.

B-Type Natriuretic Peptide Receptor Expression and Activity Are Hormonally Regulated in Rat Ovarian Cells* ALFRED NOUBANI, RIAZ FAROOKHI,

AND

JOLANTA GUTKOWSKA

Department of Medicine, Division of Experimental Medicine, McGill University (A.N., J.G.); the Laboratory of Cardiovascular Biochemistry, CHUM-Campus Hotel-Dieu, Department of Medicine, University of Montreal (A.N., J.G.)†; and the Department of Physiology and Obstetrics and Gynecology, Royal Victoria Hospital, McGill University (R.F.)†, Montreál, Que´bec, Canada H2W 1T8 ABSTRACT Natriuretic peptides form a family of structurally related peptides known to regulate salt and water homeostasis and to cause vasodilation. Synthesis of atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides occurs mainly in the heart and brain and has been identified recently in the female reproductive tract. The expression of ANP and CNP as well as their cognate guanylyl cyclase receptors (NPR-A and NPR-B, respectively) have been detected in the rat ovary. We have shown previously that the expression of the natriuretic peptides and their receptors in the rat ovary appears to be modulated by the estrous cycle. In the present study we have evaluated the expression of the natriuretic peptide system (peptide and receptor) in ovarian cells (granulosa and thecal-interstitial cells) obtained from immature female rats treated with either diethylstilbestrol (DES), an estrogen analog, or equine CG (eCG), a gonadotropin that possesses both LH and FSH activity. Using a whole cell RRA, we found that CNP binding was increased

by 2-fold in granulosa cells taken from animals treated with either DES or eCG. Semiquantitative RT-PCR revealed that granulosa cells from DES- or eCG-treated animals have increased levels of NPR-B messenger RNA (mRNA) transcripts, which was in good agreement with the increased binding. The activity of the receptors was assessed by ligand-dependent stimulation of cGMP release. CNP, but not ANP, stimulated the release of cGMP from granulosa cells obtained from DES-treated, but not from eCG-treated, animals. The relative levels of CNP mRNA in granulosa cells were unaltered by either DES or eCG treatment. In contrast, CNP mRNA levels were increased more than 2-fold, but only in theca-interstitial from the eCG-treated animals. Our results indicate that CNP and NPR-B are expressed in the ovary, and their expression is responsive to hormonal treatments. Furthermore, expression of these components of the natriuretic peptide system appears to be compartmentalized, with CNP being derived from the extrafollicular compartment and acting, through NPR-B, on the granulosa cells. (Endocrinology 141: 551–559, 2000)

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ATRIURETIC PEPTIDES form a family of structurally related peptides. Their properties include modulation of water and salt homeostasis (1), vasorelaxation (2), and regulation of cell proliferation and differentiation (3, 4). Three forms of natriuretic peptides, termed atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides, have been described and shown to arise from distinct genes (5, 6). Although these peptides share a similar primary structure (all three peptides are characterized by a 17-amino acid ring linked by a disulfide bond), their natriuretic and vasoactive properties differ (7). The major site of ANP synthesis is in the heart atrium (1), whereas BNP synthesis occurs mainly in the ventricle [although it was first identified in porcine brain (8)]. CNP was isolated originally from porcine brain (9), but it is commonly referred to as a vascular hormone and has potent vasoactive properties but little natriuretic activity (10). Natriuretic peptides have been found in other tissues besides the heart, brain, and vasculature (11). Protein or messenger RNA (mRNA) transcripts for the different natriuretic peptides have been localized to the kidney, adrenal glands, and male and female reproductive tracts (12–14).

Natriuretic peptides exert their effects by binding to their cognate membrane receptors (NPRs). Three receptor forms have been identified and are detected in a number of tissues (7). Two of these receptor forms, termed NPR-A and NPR-B, are signal-transducing, single pass, transmembrane glycoproteins exhibiting ligand-dependent intrinsic guanylyl cyclase activity (15, 16). ANP and BNP have been shown to bind preferentially to NPR-A, whereas CNP displays a greater affinity for NPR-B (17). The relative selectivity of these receptors suggests that their differential tissue expression may allow for selective activation. The glycosylation and phosphorylation status of NPR-A and NPR-B has been shown to affect ligand binding and cyclase activity (18 –20). The third receptor form, NPR-C, lacks the cyclase domain and binds all peptides with equal affinity. NPR-C has been postulated to act as a clearance receptor (21). In a previous study (14), we demonstrated that components of the ANP system are expressed in the rat ovary. Subsequent autoradiographic studies (22) suggested that both ANP and CNP bind to follicular granulosa cells. Whole ovarian radioimaging suggested that there were changes in the relative binding of these peptides accompanying the phases of the estrous cycle. To address the possibility that expression of the natriuretic peptide system in the ovary may be under either sex steroid or gonadotropin control and to resolve whether the observed binding corresponded to functional forms of NPR-A and NPR-B receptors, we examined the effects of estrogen and gonadotropin treatment of im-

Received June 11, 1999. Address all correspondence and requests for reprints to: Jolanta Gutkowska, Ph.D., Laboratory of Cardiovascular Biochemistry, Hoˆtel-Dieu du CHUM, 3850 St. Urbain Street, Pavillon Masson, Montreál, Que´bec, Canada H2W 1T8. E-mail: [email protected]. * This work was supported by funds from the Medical Research Council of Canada (to R.F. and J.G.). † Both laboratories contributed equally to this study.

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OVARIAN NATRIURETIC PEPTIDE SYSTEM

mature female rats on expression of the natriuretic peptides and their receptors in isolated granulosa and thecal-interstitial cells. Immature rats were used because hormonal treatments of these animals provides well defined stages of follicular development without the attendant presence of postovulatory structures, i.e. corpora lutea. Isolated granulosa and thecal-interstitial cells were examined for radiolabeled ANP and CNP binding. Expression of mRNA transcripts for the natriuretic peptides and receptors were assessed using semiquantitative RT-PCR. The activity of the natriuretic peptide receptors on these cells was further tested by evaluating cGMP release in response to either ANP or CNP stimulation. Our results indicate that CNP and NPR-B are the major active natriuretic peptide system in the rat ovary. Furthermore, we found that both estrogen and gonadotropin exert differential effects on the expression of this natriuretic peptide and its receptor in ovarian cells. Materials and Methods Animals All animal treatments and procedures were conducted in accordance with the institutional animal care protocols and complied with the regulations set by the Canadian Council for Animal Care. Immature (21-day-old) Sprague Dawley female rats were purchased from Charles River Laboratories, Inc. (St. Constant, Canada). They were housed under a 12-h light, 12-h dark cycle and provided with water and rat chow ad libitum. Estrogen treatment was attained by the insertion (sc) of SILASTIC brand (Dow Corning Corp., Midland, MI) implants containing diethylstilbestrol (DES; Steraloids, Wilton, NH), a potent estrogen analog. Implants were prepared as described by Sanders and Midgley (23). Animals were killed, by decapitation, 72 h after implant insertion. Gonadotropin stimulation was attained by injection (sc) of 20 IU equine CG (eCG; Ayerst, Montreal, Canada) dissolved in 200 ␮l PBS. These animals were killed 48 h after injection. Control animals were injected with an equivalent volume (200 ␮l) of PBS. The treatments were staggered to allow tissue collection from all treatment groups on the same day. The ovaries were removed and cleared of adherent tissue, and the granulosa cells were expressed as described previously (24). The thecalinterstitial ovarian tissue, now relatively devoid of granulosa cells, was homogenized briefly and passed through an 18-gauge needle. The granulosa and thecal-interstitial cell preparations were resuspended (at 107 cells/ml) in either cold PBS with 0.1% gelatin and 0.01% methiolate or in cold DMEM and stored on ice until used. Aliquots (1 ml) of the cells from control, DES-treated, and eCG-treated animals were removed, pelleted by brief centrifugation (13,000 ⫻ g for 30 sec), and frozen in liquid nitrogen for later RNA extraction. For the studies involving cGMP stimulation, six animals from each treatment group were killed, and the granulosa and thecal-interstitial cells obtained from the ovaries of two animals were pooled and placed in DMEM.

RRAs Rat ANP, CNP, and C-ANF were purchased from Peninsula Laboratories, Inc. (Belmont, CA). C-ANF is a synthetic five-amino acid ring-deleted ANP analog that binds specifically to clearance receptors (21). All other reagents were purchased from Sigma (Oakville, Canada) unless specified otherwise. 125I-Labeled rat ANP and CNP were prepared and purified by HPLC as described previously (14). The specific activities of these preparations, determined using a double displacement assay (25, 26), were 39 ␮Ci/␮g for ANP and 53 ␮Ci/␮g for CNP. Binding assays were conducted by incubating 100 ␮l (106 cells) of the granulosa or thecal-interstitial cell suspension with 50 ␮l binding buffer (50 mm Tris-HCl buffer, pH 7.4, containing 100 mm NaCl, 5 mm MgCl2, 25 mm MnCl2, 1 mm EDTA, 0.1% bacitracin, 0.5 mm phenylmethylsulfonylfluoride, and 0.4% BSA) with or without unlabeled ligand at a concentration of 10⫺6 m and the appropriate labeled peptide (⬃50,000 cpm/50 ␮l). The assay tubes were incubated at 25 C for 90 min, after which the incubates were diluted with 3 ml ice-cold 50 mm Tris-HCl buffer (pH 7.4) and immediately filtered through Whatman GF/C filters (VWR Scientific, Mont Royal, Canada) presoaked in 1% polyethylenimine. The filters were washed twice with 3 ml Tris-HCl buffer and dried, and the associated radioactivity was determined using a Cobra II ␥-counter (Canberra-Packard, Downers Grove, IL). Specific binding was determined as the difference between binding in the absence and presence of 10⫺6 m unlabeled peptide. Competition assays were conducted in a similar fashion, except that unlabeled peptides at concentrations ranging between 10⫺12–10⫺6 m were used.

RNA extraction and complementary DNA (cDNA) synthesis Total RNA from granulosa or thecal-interstitial cells was extracted using the acid guanidium-thiocyanate-phenol-chloroform method (27). RNA integrity was verified by gel electrophoresis, and RNA concentrations were assessed by spectrophotometry. RNA (1 ␮g) was reverse transcribed using random primers (Pharmacia, Montreál, Que´bec, Canada) and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Burlington, Canada), according to the manufacturer’s instructions.

Semiquantitative PCR The cDNA obtained was subjected to PCR amplification using the primer sets and conditions described in Table 1. PCR was conducted in a reaction volume of 50 ␮l containing 2 ␮l cDNA, 40 pmol of the appropriate primer set, 2.5 U Taq DNA polymerase (Life Technologies, Inc.), 10 ␮Ci [␣-32P]deoxy (d)-CTP (Amersham Pharmacia Biotech, Oakville, Canada), 1 ␮l dNTPs (10 mm dATP, dCTP, dGTP, and dTTP), 1.5 mm MgCl, 5 ␮l Taq buffer (Life Technologies, Inc.), and 34 ␮l deoxyribonuclease-free water. To determine the linear amplification conditions, PCR reactions were conducted for all of the primer sets. Aliquots (10 ␮l) were taken every two cycles starting at cycle 16. The aliquots were loaded onto a 1.5% agarose gel and electrophoresed at 70 mV for 1.5–2.0 h. The resolved products were vacuum transferred to Amersham Pharmacia Biotech Nylon H⫹ membranes (Amersham Pharmacia Biotech), and the membranes were placed in a phosphor-sensitive cassette. The radioactive bands were visualized by scanning the cassette in a phosphorimager and quantitated using ImageQuant software (Molecular Dynamics, Inc.,

TABLE 1. PCR primer sequences and amplification conditions Conditions for linear amplification Gene

CNP Sense Antisense NPR-B Sense Antisense GAPDH Sense Antisense

Primer sequence

Annealing temperature (C)

Cycle no.

5⬘-CGGCACCATGCACCTCTCCCAGCTGAT 5⬘-CAGGAGTCCAGGAGGTCCTTTTCG

63

21

5⬘-GCTACATGGTACCACCATATTGGACAACCTC 5⬘-CAGGAGTCCAGGAGGTCCTTTTCG

65

26

5⬘-AGGTCGGTGTCAACGGCTTTGGCCGT 5⬘-CAGTGCTGGCATGGACTGTGGTC

63

21


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Sunnyvale, CA). The intensity units were graphed, and the cycle number, within the linear range of the curve, was chosen for subsequent semiquantitation. For semiquantitative RT-PCR, the reactions were conducted as described above (using the linear amplification conditions), and the levels of amplified product were normalized to their corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Adjusting the saline-treated values to represent 100%, the hormonetreated values were compared with the saline values and plotted as a percentage of the control.

cGMP stimulation The effects of ANP and CNP on cGMP release into the medium were measured in cell incubates. For each treatment group (saline controls, DES-treated, and eCG-treated animals) and cell preparation (granulosa and thecal-interstitial), three separate incubations were conducted: unstimulated, i.e. buffer alone (containing 1 mm acetic acid, which was used as a solvent for the preparation of peptide stock solutions), ANP (10⫺7 m), and CNP (10⫺7 m). Details of this stimulation procedure have been described previously (14). Briefly, cell aliquots in medium (⬃106 cells in 500 ␮l DMEM) containing 0.5 mm isobutylmetylxanthine were incubated with buffer, ANP, or CNP. The tubes were placed on a shaker inside a CO2 incubator and shaken at 180 rpm for 3 h. At the end of this period, the cell suspensions were transferred to 1.5-ml microtubes and centrifuged (5 min, 13,000 ⫻ g). The supernatant was removed and frozen for later determination of cGMP content by RIA (15). The cell pellet was resuspended in 100 ␮l water, sonicated, and assayed for protein content using the Pharmacia protein reagent (Pharmacia Biotech, Montreal, Canada). The dose-response data were generated as described above, except that stimulation of the cell preparations was conducted using varying natriuretic peptide concentrations (10⫺12–10⫺7 m).

Statistical analysis All data are represented as the mean ⫾ sem. All experiments were repeated at least three times, using three to five separate samples for each treatment group. Data were analyzed by ANOVA, and multiple group comparisons were assessed using the least significant difference test. Differences between treatment groups were considered significant at P ⱕ 0.05.

Results Ovarian cells express NPR-B

Figure 1 illustrates the binding of [125I]Tyr°-CNP to preparations of granulosa (A) or thecal-interstitial (B) cells in the presence of increasing concentrations of unlabeled CNP, ANP, or C-ANF. The cell preparations were derived from DES-primed animals, as our studies (see below) indicated that labeled CNP binding was enhanced in ovarian cells derived from these animals. Although both CNP and ANP were effective in competing with [125I]Tyr°-CNP binding to these preparations, clearly CNP was the better competitor. Fifty percent displacement of the labeled peptide was achieved with approximately 10⫺9 m CNP, but a 100-fold higher concentration of ANP (10⫺7 m) was required to achieve the same degree of displacement. C-ANF, even at the highest concentration tested (10⫺6 m) was virtually ineffective in displacing [125I]Tyr°-CNP binding to these preparations. These results suggest that rat ovarian cells express only the NPR-B receptor form. To confirm this possibility, we repeated these studies using [125I]ANP as the labeled ligand. In contrast to our findings using labeled CNP, only low levels of [125I]ANP binding to these preparations were observed (Fig. 2). Although CNP was able to displace [125I]ANP, there was only 25% displacement in both granulosa and thecalinterstitial cells. ANP, but not C-ANF, displaced [125I]ANP at 10⫺6 m, to the same extent as CNP (Fig. 2, A and B). These

FIG. 1. Displacement curves for [125I]Tyr°-CNP in ovarian cells. Specific binding of [125I]Tyr°-CNP in granulosa cells (A) and thecal-interstitial (B) derived from immature female rats treated with DES. Competition of the labeled ligand with unlabeled CNP (closed circles), ANP (open circles), and C-ANF (open triangles) at concentrations ranging from 10⫺12–10⫺6 M is shown. Values are represented as a percentage of total specific binding in the absence of unlabeled peptide. These data plotted are from three separate experiments.

observations suggest that ANP binds weakly to NPR-B and that binding of radiolabeled ANP was nonspecific. Collectively, these data indicate that ovarian cells express the NPR-B form of natriuretic receptors and that either the NPR-A form is not present or these receptor levels are too low to be detected by this assay. The absence of competition by C-ANF suggests that these tissues do not possess the clearance form of natriuretic receptors. Hence, subsequent binding studies were conducted using [125I]Tyr°-CNP only. CNP binding is enhanced in granulosa cells, but not in thecal-interstitial cells, by DES and eCG treatment

The effects of DES or eCG treatments on CNP binding to granulosa and thecal-interstitial cells are illustrated in Fig. 3. DES and eCG treatments resulted in a significant increase (⬃2-fold) in CNP binding to granulosa cells compared with saline-treated controls (Fig. 3A). In contrast, neither treatment significantly altered the level of CNP binding in the

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relative levels of NPR-B transcripts approximately 2-fold in granulosa cells compared with cells derived from the salinetreated controls. No significant effects of these treatments on NPR-B mRNA levels were observed for the thecal-interstitial cells (Fig. 5B). It is noteworthy that the increase in the relative NPR-B mRNA levels in the granulosa cells of DES and eCGtreated animals is in good agreement with the increased binding levels seen in these cells (Fig. 3). We also evaluated whether NPR-A mRNA levels were affected by these treatments. Although NPR-A transcripts were detected in the total RNA isolated from granulosa and thecal-interstitial cells, these levels were unaffected by hormone treatment of the animals (data not shown). CNP stimulates cGMP release in granulosa cells

To determine whether the increased CNP binding seen in granulosa cells has functional significance, CNP-stimulated cGMP release by these cells was assessed. Figures 6 and 7 illustrates the dose-response curves for ANP and CNP-stimulated cGMP release, respectively, in granulosa (A) and thecal-interstitial (B) cells from DES-treated animals. ANP did not stimulate cGMP release even at the highest dose of 10⫺7 m in both granulosa and thecal-interstitial cells. CNP, however, did stimulate cGMP release from granulosa cells from DES-treated animals (Fig. 7A). In contrast, thecal-interstitial cells showed a slight, but insignificant, CNP-stimulated cGMP release. Figure 8 demonstrates ANP- and CNP-stimulated release of cGMP from granulosa and thecal-interstitial

FIG. 2. Displacement curves for [125I]ANP in ovarian cells. Specific binding of [125I]ANP in granulosa cells (A) and thecal-interstitial cells (B) derived from immature female rats treated with DES. Competition of the labeled ligand with unlabeled CNP (closed circles), ANP (open circles), and C-ANF (open triangles) at concentrations ranging from 10⫺12–10⫺6 M is shown. Values are represented as a percentage of total specific binding in the absence of unlabeled peptide. These values represent the data from one experiment.

thecal-interstitial cells compared with the binding observed for the saline controls (Fig. 3B). NPR-B mRNA expression is increased in granulosa cells, but not in thecal-interstitial cells, by DES and eCG treatment

To determine whether the increased CNP binding in granulosa cells after DES or eCG treatment resulted from increased NPR-B gene expression, mRNA levels for NPR-B were assessed using semiquantitative RT-PCR. Figure 4 illustrates the intensities of the radioactive bands for CNP, NPR-B, and GAPDH with respect to amplification cycle number in granulosa cells from DES-treated animals. These data clearly show that the cycle number used in the semiquantitative RT-PCR (CNP, 21 cycles; NPR-B, 26 cycles; GAPDH, 21 cycles) is within the linear range of amplification. As depicted in Fig. 5A, both treatments increased the

FIG. 3. Specific binding of [125I]Tyr°-CNP to granulosa (A) and thecalinterstitial (B) cells derived from saline-treated (open bar), DES-treated (closed bar), and eCG-treated rats (hatched bar). Binding is expressed as micrograms of CNP bound per ␮g DNA and represent the mean ⫾ SEM for tissue derived from four rats in each treatment group. *, Significant difference (P ⬍ 0.05) from saline-treated controls (n ⫽ 3).


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FIG. 4. Phosphorimage analysis of RT-PCR amplification using specific oligonucleotide primers for CNP (top panel), NPR-B (middle panel), and GAPDH (lower panel). Total RNA from granulosa cells of DES-treated rats was reverse transcribed, and the cDNA was amplified using increasing number of cycles. The relative intensity units were plotted, and the cycle number for each primer set was established as being within the linear range of amplification. This image is a representative of two separate experiments (n ⫽ 2).

cells from saline-, DES-, and eCG-treated animals. CNP (10⫺7 m) stimulated cGMP release by approximately 2-fold in granulosa cells from DES-treated animals. In contrast, no significant increase in cGMP release was seen when granulosa cells from eCG-treated animals were similarly stimulated (Fig. 8A). No significant effects on thecal-interstitial stimulation with CNP were observed (Fig. 8B) regardless of the prior hormonal treatment of the animals. The basal (unstimulated) levels of cGMP secreted by thecal-interstitial cells, however, were considerably higher than the levels observed for granulosa cells. ANP (10⫺7 m) had no significant effect on cGMP secretion from either tissue, although cGMP levels were slightly elevated by ANP in granulosa cells from the salinetreated controls. eCG treatment increases CNP mRNA expression in thecalinterstitial cells

The effects of hormone treatment on the levels of CNP mRNA levels in granulosa and thecal-interstitial cells were assessed by semiquantitative RT-PCR. The relative levels of CNP mRNA transcripts in granulosa cells from DES- or eCG-treated animals were decreased compared with the levels in saline-treated animals (Fig. 9A). This decrease, however, was not significant. CNP mRNA levels were unaffected in thecal-interstitial cells from DES-treated animals (Fig. 9B). In contrast, thecal-interstitial cells from eCG-treated animals

FIG. 5. Semiquantitative RT-PCR analysis of NPR-B mRNA levels in granulosa cells (A) and thecal-interstitial cells (B) derived from saline-treated (open bar), DES-treated (closed bar), and eCG-treated (hatched bar) rats. Total RNA from immature rats was reverse transcribed, and the cDNA was amplified using PCR primers specific for NPR-B or GAPDH. The NPR-B to GAPDH ratio for the saline-treated animals was set at 100%. The data represent the mean ⫾ SEM for tissue derived from four rats in each treatment group. *, Significant difference (P ⬍ 0.05) from saline-treated controls (n ⫽ 3). Below each histogram is the corresponding phosphorimage scan of the PCR products detected using the primers labeled to the left.

showed a significant increase of approximately 2.5-fold in CNP mRNA levels (Fig. 9B). Discussion

The development and maturation of ovarian follicles are regulated primarily by the gonadotropins FSH and LH (28). A number of studies have shown, however, that additional local regulatory processes are evoked by the gonadotropins through their effects on the synthesis and secretion of intraovarian factors or by their modulation of the receptors for these factors (29). A striking feature of this intraovarian regulatory network is that it is compartmentalized, as it reflects the separate primary targets for the gonadotropins, i.e. the theca-interstitium, which is responsive to LH, and the gran-

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FIG. 6. Dose-response curves for ANP-stimulated cGMP release from granulosa (A) and thecal-interstitial (B) cells derived from DEStreated animals. Cells were incubated with increasing concentrations of ANP (10⫺12–10⫺7 M) for 3 h, after which the media were recovered, and cGMP content was measured by RIA. cGMP content is expressed as picomoles of cGMP released per mg protein and represent the mean ⫾ SEM for tissue derived from three separate experiments using two rats for each concentration. *, Significant difference (P ⬍ 0.05) from 10⫺12 M (n ⫽ 3).

ulosa cell compartment, which is responsive to FSH (30). The results of our present study indicate that the natriuretic peptide system may be an important component of this intraovarian regulatory network. Our results identify CNP and its cognate receptor, NPR-B, as prominent components of the natriuretic peptide system in the rat ovary. Our results clearly demonstrate that both CNP and NPR-B expression in the rat ovary can be modulated by hormonal treatments. Furthermore, these results indicate that expression of the ligand (CNP) and its receptor (NPR-B) are compartmentalized within the ovary. Our results point to CNP derivation from the thecal-interstitial compartment (theca-interstitium), whereas the granulosa cells appear to be the target for this peptide. A specific role for CNP in the ovary, however, was not examined in this study and remains to be elucidated. We have identified NPR-B as the major natriuretic peptide receptor type in the rat ovary. Hormonal modulation of this receptor, however, appears to be limited to the granulosa cells. Both estrogen and eCG treatments increase CNP binding to the same extent in granulosa cells. As eCG treatment of immature rats will stimulate endogenous estrogen production in granulosa cells (31), we suspect that the observed increase in the

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FIG. 7. Dose-response curves for CNP-stimulated cGMP release from granulosa (A) and thecal-interstitial (B) cells derived from DEStreated animals. Cells were incubated with increasing concentrations of ANP (10⫺12–10⫺7 M) for 3 h, after which the media were recovered, and cGMP content was measured by RIA. cGMP content is expressed as picomoles of cGMP released per mg protein and represents the mean ⫾ SEM for tissue derived from three separate experiments using two rats for each concentration. *, Significant difference (P ⬍ 0.05) from 10⫺12 M (n ⫽ 3).

eCG-treated animals arises from this endogenous estrogen signal. As neither treatment affected thecal-interstitial CNP binding, we conclude that either this tissue exhibits a constitutive low level of CNP binding or the binding observed is due to a small degree of granulosa contamination of the thecal-interstitial preparation. We suspect the latter possibility, because our previous autoradiography studies indicated pronounced binding to the granulosa cells and little binding to thecal-interstitial cells (22). Furthermore, only granulosa cells responded to CNP stimulation with an increase in cGMP secretion. The possibility that the thecal-interstitial cell compartment also expresses some NPR-B cannot be rejected outright. Ongoing studies, using preparations of purified thecal-interstitial cells, may help to clarify this issue. We found excellent agreement between the degree of enhanced CNP binding and the relative increase in NPR-B mRNA levels in granulosa cells from the DES- and eCG-treated animals. This suggests that expression of this receptor is at least in part under transcriptional regulation. Estrogen, whether it is exogenously supplied or its production endogenously stimulated, appears to be involved in this transcriptional regulation. Interestingly, estrogen increases NPR-A activity in PC-12 cells


FIG. 8. ANP- and CNP-stimulated cGMP release from granulosa (A) and thecal-interstitial (B) cells derived from saline-, DES-, and eCGtreated animals. Cells were incubated with buffer (open bar), ANP (10⫺7 M; hatched bar), or CNP (10⫺7 M; closed bar) for 3 h, after which the media were recovered, and cGMP content was measured by RIA. cGMP content is expressed as picomoles of cGMP released per mg protein and represents the mean ⫾ SEM for tissue derived from three separate experiments using three rats for each treatment group. *, Significant difference (P ⬍ 0.05) from buffer-treated controls (n ⫽ 3).

(32) and rat adrenal zona glomerulosa cells (33). As mentioned previously, NPR-A and NPR-B levels in the rat uterus are also increased after treatment with this steroid. Collectively, these observations strongly implicate estrogen as an important regulator of natriuretic peptide receptors in a number of cell and tissue types. There is evidence that estrogen can also enhance natriuretic peptide expression. Hong et al. (34) showed that ANP mRNA levels were increased in the atria of estrogentreated ovariectomized rats. We did not, however, observe an effect of estrogen on either ANP (data not shown) or CNP mRNA expression in either granulosa or in thecal-interstitial cells. Thus, at least in the case of the rat ovary, the actions of estrogen appear to be limited to natriuretic peptide receptor regulation. The presence of NPR-A mRNA transcripts in granulosa and thecal-interstitial cells is puzzling, because we could not demonstrate any specific ANP binding in either of these preparations. This observation suggests that expression of the NPR-A gene does not lead to detectable levels of functional receptor. This does not agree with our previous observation that ANP binds to and stimulates cGMP production in preparations derived from ovaries of adult cycling rats (14). In the present study, however, the cells were derived from the ovaries of

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FIG. 9. Semiquantitative RT-PCR analysis of CNP mRNA levels in granulosa (A) and thecal-interstitial (B) cells derived from salinetreated (open bar), DES-treated (closed bar), and eCG-treated (hatched bar) rats. Total RNA from immature rats was reverse transcribed, and the cDNA was amplified using PCR primers specific for NPR-B or GAPDH. The NPR-B to GAPDH ratio for the saline-treated animals was set at 100%. The data represent the mean ⫾ SEM for tissue derived from four rats in each treatment group. *, Significant difference (P ⬍ 0.05) from saline-treated controls (n ⫽ 3). Below each histogram is the corresponding phosphorimage scan of the PCR products detected using the primers labeled to the left.

immature animals. These ovaries, therefore, would be devoid of luteinized cells. We suspect that the pronounced ANP binding and cGMP stimulation previously observed arise from the action of ANP on luteinized cells. Pandey et al. (35) have shown that ANP stimulates steroidogenesis in human granulosa-lutein cells. We have preliminary evidence indicating that ovarian preparations derived from luteinized ovaries of immature rats display ANP binding. The ability of ANP to compete with CNP binding to NPR-B has been shown in several systems (7, 17), although in all cases high concentrations of ANP were required. A similar situation appears to exist in ovarian cells. What is surprising, however, is that we could not detect any specific binding of radiolabeled ANP to these preparations. This would suggest that NPR-B is exquisitely specific for CNP and that the binding of ANP to NPR-B must be transient. The specificity of NPR-B for CNP is further emphasized by our assessment of cGMP stimulation from granulosa cells. Only CNP was able to stimulate, significantly, cGMP release in granulosa cells from saline- or estrogen-treated rats. The absence of cGMP

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stimulation but retention of CNP binding to granulosa cells from eCG-treated animals indicate that these cells have desensitized their response to CNP. This phenomenon has been demonstrated for NPR-A, where activation of protein kinase C resulted in receptor dephosphorylation and the loss of cyclase activation, but left ligand binding unaffected (19). Potter (18) showed that NPR-B can be desensitized by exposure to CNP, and this appears to involve dephosphorylation of the receptor. The latter observation is particularly intriguing, as our results suggest that CNP mRNA (and presumably peptide) is up-regulated by eCG treatment in thecal-interstitial cells. This increased CNP may be responsible for the desensitization of the granulosa cell receptors. If this is the case, it would indicate that gonadotropins may exert bimodal effects on CNP signaling in the ovary. The initial action of gonadotropins would promote estrogen synthesis, which would promote granulosa NPR-B expression and CNP responsiveness, whereas subsequent gonadotropin action would curtail this response. As we observed increases in CNP mRNA expression in thecal-interstitial cells only, this leads to the inference that CNP expression is under LH control. The continued increases observed in circulating LH concentrations during the period leading up to the LH surge would be consistent with the above scenario. Furthermore, this scenario identifies a window during which CNP would be expected to act, i.e. during antral follicular development. A role for natriuretic peptides in the ovary remains to be defined. Sandberg et al. (36) demonstrated that ANP enhanced progesterone-induced maturation of Xenopus oocytes. To¨rnell et al. (37) showed that ANP inhibited spontaneous oocyte maturation in cumulus-oocyte complexes. These observations raise the possibility that natriuretic action may be limited to a subset of granulosa cells, i.e. cumulus cells. It should be noted, however, that the effects of CNP were not examined in either of these studies. Hence, the possibility that these effects of ANP are exerted through NPR-B cannot be discounted, although our studies would indicate that this is unlikely. In any event, these observations suggest that natriuretic peptides may be important in modulating aspects of oocyte maturation, a possibility that needs to be explored in more detail. A possible physiological role for CNP in the ovary could be an ability to modulate follicular atresia (apoptosis). McGee et al. (38) have shown that the addition of 8-bromocGMP can dramatically decrease the follicular atresia observed for follicles grown under serum-free conditions in the presence of gonadotropins or cAMP. Follicular atresia is a dominant event in the ovary and appears to be most pronounced for follicles at the preantral to antral transition (39). Appropriate CNP and NPR-B expression in follicles at these stages may, therefore, through stimulation of cGMP, be involved in this follicle selection process. In summary, we have provided evidence that the natriuretic peptide system is expressed in the ovary and is modulated by hormones in this organ. Estrogen and gonadotropin are the important regulators of this system and allow for yet another example of the complex interactions and communication seen between follicular compartments.

Acknowledgments The authors thank Naomi Machell for helpful discussion and suggestions. The patience and technical assistance of Ce´line Coderre and Nathalie Charron are much appreciated. The authors thank Dominique Poutrieux for her excellent and efficient preparation of the manuscript.

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International Course on Laboratory Animal Science—Utrecht, The Netherlands May 15–26, 2000 A two-week intensive course on laboratory animal science will be organized at the Department of Laboratory Animal Science—Utrecht, The Netherlands, in May 2000. This course has been organized once a year since 1993. The objective of this course is to present basic facts and principles that are essential for the humane use of animals and for the quality of research. The contents of the course are in line with recommendations of the Federation of European Laboratory Animal Science Associations (FELASA) regarding the training of the young scientist whose research involves the use of vertebrate animals. The course may also be of interest for those who intend to set up a similar course at their location. For this purpose, during the course the acquisition of teaching materials can be discussed with the course committee. For information and application forms please contact: Prof. Dr. L. F. M. van Zutphen or Mr. Stephan van Meulebrouck, Department of Laboratory Animal Science, Faculty of Veterinary Medicine, P.O. Box 80.166, 3508 TD Utrecht, The Netherlands. Phone: 31-30-2532033; Fax: 31-30-2537997; E-mail: [email protected].