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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.)
1 ; and the Department of Physiology and Obstetrics and
Gynecology, Royal Victoria Hospital, McGill University (R.F.)
2 ,
Montréal, Québec, Canada H2W 1T8
Address all correspondence and requests for reprints to: Jolanta Gutkowska, Ph.D., Laboratory of Cardiovascular Biochemistry, Hôtel-Dieu du CHUM, 3850 St. Urbain Street, Pavillon Masson, Montréal, Québec, Canada H2W 1T8. E-mail: jolanta.gutkowska{at}umontreal.ca
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Abstract |
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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.
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Introduction |
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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, 19, 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 immature 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µCi/µg for ANP and 53
µCi/µg for CNP. Binding assays were conducted by incubating 100
µl (106 cells) of the granulosa or
thecal-interstitial cell suspension with 50 µl 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 µl). 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 µg) was
reverse transcribed using random primers (Pharmacia, Montréal,
Québec, 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 µl containing 2 µl cDNA, 40 pmol of the appropriate primer
set, 2.5 U Taq DNA polymerase (Life Technologies, Inc.), 10 µCi [
-32P]deoxy (d)-CTP
(Amersham Pharmacia Biotech, Oakville, Canada), 1 µl
dNTPs (10 mM dATP, dCTP, dGTP, and dTTP), 1.5
mM MgCl, 5 µl Taq buffer (Life Technologies, Inc.), and 34 µl deoxyribonuclease-free
water.
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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 µl
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 x
g). The supernatant was removed and frozen for later
determination of cGMP content by RIA (15). The cell pellet was
resuspended in 100 µl 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 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 thecal-interstitial 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 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.
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. 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
thecal-interstitial cells compared with the binding observed for the
saline controls (Fig. 3B).
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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 relative levels of NPR-B transcripts approximately 2-fold
in granulosa cells compared with cells derived from the saline-treated
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 eCG-treated animals is in good agreement
with the increased binding levels seen in these cells (Fig. 3
).
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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 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 saline-treated
controls.
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). 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 showed a significant
increase of approximately 2.5-fold in CNP mRNA levels (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 (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 estrogen-treated 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 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 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. Törnell 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-bromo-cGMP 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.
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Acknowledgments |
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Footnotes |
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Received June 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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