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Department of Pediatrics (L.G., A.V., M.F.) and Department of Obstetrics and Gynecology (B.F.), University of Parma, 43100 Parma, Italy; Department of Endocrinology and Metabolism (A.B., A.C.), University of Genova, 16132 Genova, Italy; Evgenidion Hospital (G.M.), Athens University, Medical School, Endocrine Unit, 11528 Athens, Greece; National Institute of Child Health and Human Development (G.P.C.), National Institutes of Health, Bethesda, Maryland 20892; and Department of Pediatrics (S.B.), University of Modena, 41100 Modena, Italy
Address all correspondence and requests for reprints to: Lucia Ghizzoni, M.D., Department of Pediatrics, University of Parma, Via Gramsci 14, 43100 Parma, Italy. E-mail: lughizzo{at}ipruniv.cce.unipr.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical
CRH9–41 antagonist at 10-6 and
10-7 mol/liter blocked the suppressive effect of
10-9 mol/liter CRH on both E2 and
P4 secretion, while it had no effect when added to the
culture media without CRH. Since interleukin (IL-1)-1 mediates certain
actions of CRH on leukocytes, we examined whether the CRH effect on
ovarian steroidogenesis was IL-1-mediated. Interleukin-1 receptor
antagonist at 10-7 and 10-6 mol/liter blocked
the inhibitory effects of CRH on E2 and P4
secretion, while it had no effect in the absence of CRH. In conclusion,
CRH exerts a CRH- and IL-1 receptor-mediated inhibitory effect on
ovarian steroidogenesis and might be actively involved in the still
enigmatic processes of follicular atresia and luteolysis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we demonstrated that rat (12) and human ovaries (13) contain
immunoreactive (Ir) CRH in theca cells surrounding follicles, as well
as in stroma cells, mature oocytes within antral follicles, and ovarian
resident macrophages. In developing corpora lutea, we detected IrCRH in
the cytoplasm of both small theca-derived and large
granulosa-derived luteinized cells, indicating that with
luteinization, CRH persists in theca-derived and appears de
novo in granulosa-derived luteinized cells. In addition, we
localized CRH receptors autoradiographically in stroma and theca cells
around follicles, as well as in cells of the cumulus oophorus. We found
CRH receptors sparsely distributed within corpora lutea but not in the
granulosa layer of follicles. By specific in situ
hybridization, Nappi and Rivest (14) confirmed these findings in rat
ovaries and characterized the ovarian CRH receptors as type I.
Ovulation, luteolysis, and, perhaps, follicular atresia, three key
ovarian functions, have characteristics of an aseptic
immune/inflammatory reaction (15). Resident macrophages constitute a
major cellular component of the ovary (16, 17), and inflammatory
cytokines, such as IL-1, IL-6, and tumor necrosis factor-,
participate in the regulation of these functions, as well as of
follicular and luteal steroidogenesis (18, 19).
Earlier, CRH and its receptors were identified in Leydig cells of the testis, where CRH was shown to exert autocrine inhibitory actions on testosterone biosynthesis (20, 21). Since the ovarian theca cell is considered the embryological and functional equivalent of the testicular Leydig cell (22), it is reasonable to hypothesize that CRH plays a similar role in the estrogen and progesterone (P4) biosynthesis of the ovary. We examined this hypothesis by measuring estradiol (E2) and P4 concentrations in the culture media of human granulosa-lutein cells incubated with ovine (o) CRH in the absence or presence of a specific CRH antagonist. In addition, to examine whether the presumed effect of CRH on ovarian steroidogenesis was IL-1 mediated, we also measured E2 and P4 concentrations in the culture media of human granulosa-lutein cells incubated with IL-1 receptor antagonist (ra) in the presence and absence of oCRH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Synthetic rat/human (r/h) CRH-(1–41) and -helical
CRH9–41 antagonist were purchased from Peninsula
Laboratories (Belmont, CA); ovine (o) CRH was obtained from UCB
(Braine-l’Alleud, Belgium); interleukin (IL)-1 receptor antagonist
(ra) (23, 24) was provided by AMGEN (Boulder, CO); HPLC-purified
[125I]r/hCRH was obtained from New England Nuclear Corp.
(Boston, MA). Multiwell dishes were purchased from Falcon Plastics (Los
Angeles, CA); medium 199 with Earle’s salt, L-glutamine,
penicillin, and streptomycin sulfate were obtained from ICN Biomedicals
(Costa Mesa, CA).
Granulosa-lutein cell cultures
After oocyte recovery, granulosa cells were obtained from
follicular fluid by centrifugation at 800 rpm for 15 min; cells were
resuspended with a 1:1 mixture of medium 199 and Hanks’ salt solution,
and pools were prepared from each individual patient. The suspension
(10 ml) was layered in 10 ml of lymphocyte separation medium (Ficoll,
ICN Biomedicals) and centrifuged at 1800 rpm at room temperature for 30
min to separate the red cells. Granulosa cells were recovered from the
Ficoll interface, washed three times with HBSS, and resuspended with 3
ml medium 199. Aliquots of the cell suspension were counted with a
hemocytometer to determine the number of cells and plated (5 x
105 cells per well) in multiwell dishes in 1 ml medium 199
with Earle’s salt supplemented with 2% L-glutamine, 1%
nonessential amino acids, 1% penicillin-streptomycin, 1% tylosin,
10% FBS. Cultures were maintained in humidified 95% air-5%
CO2 at 37 C. After 24 h, culture medium was replaced
with 1 mL serum-free medium, and cells were cultured for 24 h with
the following treatments: medium alone (control), and medium containing
oCRH and/or CRH9–41 antagonist and/or IL-1 receptor
antagonist (IL-1ra), depending on the protocol. The serum-free medium
contained 10-7 mol/liter androstenedione as substrate for
E2 production when E2 production was to be
studied. Culture media were removed after 24 h, and stored at -20
C until tested for E2 and P4 content.
Protocol
CRH effect on E2 and P4 production by
granulosa cell cultures.
The in vitro effect of CRH was
tested by adding increasing doses of oCRH (10-11 to
10-6 mol/liter) to the culture media of the granulosa
cells, which stimulates the CRH receptor as efficiently as hCRH (25).
We did not test the CRH effect with doses lower than 10-11
since such concentrations would not have provided physiologically
relevant information. Furthermore, we examined whether the CRH effect
on E2 and P4 production could be abolished by
incubating granulosa-lutein cell cultures with 10-9
mol/liter of oCRH in the presence and absence of 10-6 and
10-7 mol/liter of the -helical CRH9–41
antagonist, whose affinity for the ligand is about 5 x
10-9 M. To test the effect of the CRH
antagonist on CRH activity, we have arbitrarily chosen the
10-9 mol/liter CRH concentration as the most
physiological. IL-1ß concentrations in the culture media were also
measured at baseline and after the addition of 10-9
mol/liter CRH.
IL-1ra effect on CRH suppression of estrogen and P4
production by granulosa cell cultures.
We examined whether the
CRH-inhibitory effect on E2 and P4 production
could be abolished by incubating granulosa-lutein cell cultures with
increasing concentrations of a highly specific and selective IL-1ra
(10-7 to 10-6 mol/liter) (26, 27) with and
without 10-9 mol/liter CRH.
Each treatment was examined in at least six different experiments. Each experiment was carried out in triplicate. E2 and P4 content were measured in the media of at least six wells with granulosa cells, and these levels served as controls (basal levels) for the corresponding experiment.
Hormone assays
E2 and P4 were assayed using
commercially available RIA kits (DPC, Los Angeles, CA). The mean intra-
and interassay coefficients of variation were 5.34% and 6.4%,
respectively, for E2, and 5.39% and 6.78%, respectively,
for P4. IL-1ß was measured by the enzyme-linked
immunosorbent assay method (R&D Systems, Minneapolis, MN). The mean
intra- and interassay coefficients of variation were 2.9 and 4.9%,
respectively.
Baseline concentrations of CRH in the follicular aspirates and media of granulosa-lutein cell cultures were measured by RIA as previously described (13). The CRH antiserum (TS-2) employed has been characterized in detail previously (28). [125I]r/hCRH was used as the tracer, and synthetic r/hCRH was used as the standard. The within-assay coefficient of variation and sensitivity were 4% and 1 pmol/liter, respectively. Recovery in the CRH RIA was 82 ± 3.5%.
Statistical analysis
Data are expressed as mean ± SEM. The data
were analyzed by ANOVA followed by Scheffe’s test (29). Statistical
significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CRH inhibits E2 production by granulosa-lutein
cells (Fig. 1
)
Baseline E2 production by granulosa-lutein cells was
53.52 ± 10.88 nmol/liter (mean ± SEM). Exposure
of granulosa-lutein cells to increasing concentrations of CRH caused a
reduction of E2 concentrations in the media. E2
production was inhibited by all CRH concentrations employed between
10-10 and 10-6 mol/liter, except for the
lowest one (10-11 mol/liter). Maximal effect was obtained
at the concentration of 10-6 mol/liter, which caused a
39% decrease in E2 production. The concentrations of
10-6 or 10-7 mol/liter of the -helical
CRH9–41 antagonist blocked the suppressive effect of
10-9 mol/liter of CRH on E2 secretion by
cultured granulosa-lutein cells but had no effect when added alone to
the culture media.
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)-helical
CRH9–41 antagonist blocked the suppressive effect of
10-9 mol/liter of CRH on P4 secretion by
cultured granulosa-lutein cells but had no effect when added alone to
the culture media.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that exposure of granulosa-lutein cells to increasing
concentrations of CRH caused a moderate but consistent reduction of
both E2 and P4 concentrations in the media,
which was not dose-dependent, most probably because the system was
maximally inhibited at the lower concentration employed. This is not
surprising, since no other effect of CRH has ever been described at
levels lower than 10-11 M. For example, the
EC50 for the CRH-induced ACTH release is approximately
10-9 M. The proinflammatory effect of CRH in
uveitis (30) or caraggeenin-induced inflammation (31) starts at levels
higher than 10-11 M, as well as its inhibitory
effect on testicular steroid biosynthesis (20). The reduction of both
E2 and P4 concentrations in the media was
completely abolished by the addition of excess -helical
CRH9–41 antagonist, indicating that it is CRH
receptor-mediated. Indeed, sparse CRH-binding sites were found by
autoradiography in rat corpora lutea (12), and CRH receptors were shown
in human ovaries by in situ hybridization techniques (32).
While this study was being conducted, Calogero et al. (33)
reported a CRH-mediated inhibition of estrogen biosynthesis by FSH from
rat granulosa and human granulosa-lutein cells, which seems to be
linked to inhibition of aromatase activity. The results of the present
study are compatible with those of Calogero et al. and
further expand on the mechanisms underlying the inhibitory effect of
CRH on ovarian steroidogenesis.
In analogy to its autoregulatory action on testosterone biosynthesis in the testis (20, 21), ovarian CRH may participate in the local regulation of steroid biosynthesis in the ovaries. The inhibitory effect of CRH on E2 secretion indicates the presence of a direct and/or indirect effect of the former on the aromatase and/or 17-ketoreductase activities of the granulosa-lutein cells. The IL-1-mediated action of CRH at peripheral inflammatory sites (10) made IL-1 a possible candidate for the indirect effect of CRH in the ovary. Indeed, when granulosa-lutein cells were incubated simultaneously with CRH and IL-1ra, the CRH-induced suppression of E2 secretion was completely abolished, whereas the addition of IL-1ra alone to the cultured cells had no effect on basal E2 secretion. Furthermore, IL-1 concentrations in the culture media were significantly increased by addition of CRH, with an increment that paralleled the decrease in E2 and P4 levels induced by the same CRH concentration. These data indicate that ovarian IL-1 mediates the CRH suppressive effect on E2 secretion. IL-1 receptor is present on human granulosa cells (34), and the IL-1 gene is expressed in the gonadotropin-pretreated human ovary (35). Moreover, the IL-1ß gene is induced by forskolin (35), a potent activator of adenylate cyclase in human granulosa cells, while CRH increases intracellular cAMP in several tissues (36). Thus, ovarian CRH might activate IL-1ß production by human granulosa cells through an intracellular increase of cAMP. Alternatively, CRH might stimulate IL-1 of macrophage origin to attenuate granulosa cell aromatase activity, as previously proposed in the rat ovary (37).
The inhibitory effect of CRH on P4 secretion might also be
exerted directly and/or indirectly on different enzymatic activities.
When we incubated granulosa-lutein cells simultaneously with CRH and
IL-1ra, the CRH-induced suppression of P4 secretion was
completely abolished, whereas the addition of IL-1ra alone to the
cultured cells had no effect on basal P4 secretion. These
data indicate that the CRH suppressive effect on P4
secretion from granulosa-lutein cells is also IL-1-mediated. IL-1 has
been shown to inhibit P4 secretion in cultures of porcine
and rat granulosa cells (38), as well as androsterone production by rat
theca-interstitial cells and whole ovarian dispersates (39). The
inhibitory effect of IL-1 on gonadotropin-stimulated granulosa cell
steroidogenesis is accompanied by the arrested transcription of the
mitochondrial cholesterol side chain cleavage enzyme
(P450scc) and 3,ß-hydroxysteroid
dehydrogenase/
-5,4-isomerase (40), both of which are involved in
P4 biosynthesis. This IL-1 effect might be due to
IL-1-mediated inhibition of steroidogenic acute regulatory protein mRNA
levels (41). Steroidogenic acute regulatory protein is a mitochondrial
nonenzymatic phosphoprotein that transports cholesterol into the inner
mitochondrial membrane, a function indispensable for the acute
biosynthesis of steroids (42). Alternatively, CRH might exert a
deleterious effect on the growth of granulosa-lutein cells through its
yet unexplored proinflammatory properties, which might explain the
reduction of steroid biosynthesis via stimulation of prostanoids and
other inflammatory mediators. An association of ovarian CRH with the
aseptic inflammatory phenomena of luteolysis was suggested previously
(13).
The CRH effect on ovarian steroid biosynthesis might be associated with the elevated ovarian androgen production in women with the polycystic ovary syndrome who have decreased ovarian CRH levels (13). Conversely, ovarian CRH hypersecretion might be involved in various abnormalities of the menstrual cycle, particularly those observed during the luteal phase. Such abnormalities have been associated with anxiety and/or stress-related phenomena, which are known to be accompanied by high portal CRH levels (4). In this regard, although immunohistochemically detected ovarian CRH in corpora lutea is not grossly decreased in the rat ovary after a 2-h immobilization stress (43), CRH receptors are markedly increased (14), probably resulting in an enhanced overall biological effect of this peptide. Interestingly, in the CRH gene knock-out mouse, no major abnormality of the reproductive process was observed (44). This is not surprising, since, in this animal, other systems might have compensated for the defect, as it happened with the activity of the hypothalamic-pituitary-adrenal axis, which was not altered in a major fashion, probably because arginine vasopressin compensated for the CRH defect.
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Footnotes |
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Received March 10, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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