0013-7227/90/1275-2117$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Receptor-Mediated Actions of Growth Hormone Releasing Factor on Granulosa Cell Differentiation C. MORETTI, A. BAGNATO, N. SOLAN, G. FRAJESE, AND K. J. CATT Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD; and Clinica Medica V (G.F.), University La Sapienza, Rome, Italy
Glucagon and gastric inhibitory peptide, other peptides of the glucagon superfamily, and unrelated peptides including CRF and /3-endorphin, did not inhibit binding of either radioligand to ovarian receptors. In cultured granulosa cells, rGRF and VIP stimulated cAMP formation, consistent with coupling of their receptors to the adenylate cyclase system, and potentiated FSHinduced cAMP production. Both peptides also amplified FSHinduced progesterone biosynthesis, aromatase activity, and LH receptor formation. These observations demonstrate that rGRF is a potent cAMP-mediated agonist in the rat ovary and acts on a common VIP/GRF receptor in maturing granulosa cells. It is likely that the potentiating effect of administered GRF on gonadotropin-stimulated follicular development in vivo is in part mediated by direct actions of the peptide on the VIP/GRF receptor. Also, since GRF is present in the gonads, it is possible that the locally-produced peptide promotes follicular maturation by paracrine modulation of the stimulatory action of FSH on granulosa cell function. (Endocrinology 127: 2117-2126, 1990)
ABSTRACT. GRF promotes follicular maturation and ovulation when administered with FSH in the treatment of infertility. Such actions could be mediated by stimulation of GH secretion and insulin-like growth factor I production, but the known actions of the structurally related hormone, vasoactive intestinal peptide (VIP), on granulosa cell function suggested that GRF may also act directly on the ovary to stimulate follicular development. Radioligand binding and activation studies, performed in granulosa cells from immature estrogen-treated rats, revealed a common receptor for VIP and rat (r) GRF in the ovary. Specific binding of [125I]VIP to granulosa cells was saturable and dependent on time and temperature. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP > rGRF > peptide histidine isoleucinamide > [His\Nle27] human GRF(1-32)NH2 > secretin. In binding studies with the potent GRF agonist, [125I][His1,Nle27]GRF(l-32)NH2) relative potencies were: rGRF(l-43)OH > [HisSNle^Jhuman GRF(132)NH2 > VIP > peptide histidine isoleucinamide > secretin.
T
HE 43 amino acid hypothalamic polypeptide, GRF, is the major physiological stimulus of GH secretion from the anterior pituitary gland (1). In the rat, GRF was isolated from hypothalamic extracts on the basis of its ability to stimulate GH secretion from cultured anterior pituitary cells (2). GRF-like immunoreactivity has since been found in several extrahypothalamic sites (35), including the gastrointestinal tract (6, 7), human tumors (8, 9), tumors of neuroendocrine origin (10), and placental extracts (11-13), suggesting that GRF may also have a regulatory role in several peripheral tissues. At the gonadal level, a GRF-like substance and its messenger RNA (mRNA) have been identified in the postpuberal rat testis (14). In man, GRF-like material has been demonstrated in both ovarian and in testicular tissue by the immunoperoxidase technique (15), and immunoreactive GRF has been detected in ovarian follicular fluid Received July 18, 1990. Address requests for reprints to: Dr. Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 10, Room B1-L400, Bethesda, Maryland 20892.
(16). In infertile women resistant to gonadotropin therapy, the administration of GRF(l-29) in association with pure FSH activates folliculogenesis and stimulates the development of a dominant ovarian follicle (16). The amino acid sequence of rat (r) GRF has striking homologies with the sequence of the vasoactive intestinal peptide (VIP) peptide histidine isoleucinamide (PHI)/ secretin/Helodermin/Helospectin family of peptides (17, 18). Complementary DNA clones which encode the precursor to VIP and GRF have been isolated and, despite the distinction in size and sequence of the prepropeptides, the structural organization of the two precursors is similar (19). The homology between these two peptides also extends to their ability to elicit biological responses in several target tissues. The broad range of actions of VIP/PHI in various target cells includes alterations in water and electrolyte transfer in epithelial cells (20), stimulation of glycogenolysis in hepatocytes (21), and relaxation of smooth muscle (22). Specific recognition sites for VIP are present in rat liver (23) fat (24), intestine (25), brain (26), lung (27), pancreas (28), and prostate (29). In the brain and the adrenal gland, ACTH,
2117
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2118
GRF ACTIONS ON GRANULOSA CELLS
VIP, GRF, and dynorphin compete for common receptors (30). In the rat pituitary gland, receptors for VIP have been characterized on lactotropes (31). There is considerable evidence for a role of VIP in the control of reproductive function. Immunoreactive VIP and VIPergic nerve fibers are present in the female genital tract of animals (32) and humans (33), in association with the smooth muscle of blood vessels and in the ovarian stroma (34). VIP stimulates steroidogenesis in cultured rat granulosa cells (35), regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex which catalyzes the rate-limiting reaction in progesterone biosynthesis (36), and enhances aromatase activity in the neonatal rat ovary before the development of primary follicles, at a stage in which the follicles are not yet responsive to FSH (37). VIP also stimulates oocyte maturation and cAMP production in isolated preovulatory rat follicles (38), and plasminogen activator activity in avian granulosa cells (39). VIP-encoding mRNA has been detected in rat ovaries, suggesting local synthesis of the peptide (40). The present study was performed to analyze the interaction between GRF and the VIP receptor in rat granulosa cells. To this end, we characterized the specific binding of [125I]VIP and [125I][His\Nle27] human (h) GRF(1-32)NH2, a potent analog of rGRF (41), to these cells and compared the actions of these structurally related peptides on gonadotropin-induced differentiation and steroidogenesis in rat granulosa cells. These studies have revealed potent synergistic actions of both rGRF and VIP on FSH-induced granulosa cell maturation.
Endo • 1990 Vol 127 • No 5
diol were supplied by Hazleton Biotechnologies (Vienna, VA). 3-Isobutyl-l-methylxanthine (MIX) was purchased from Aidrich (Milwaukee, WI). Preparation of granulosa cells
Granulosa cells were prepared from ovaries of estrogentreated immature female rats as previously described (42). Silastic capsules (10 mm) containing diethylstilbestrol were implanted sc on day 21 to stimulate granulosa cell proliferation, and animals were killed between the ages of 25-32 days by decapitation. Ovaries were removed under aseptic conditions and dissected free of adherent connective tissue in sterile modified McCoy's 5a medium supplemented with 10 mM HEPES, pH 7.4, 4 mM L-glutamine, 100 U/ml penicillin, and 100 iig/rcA streptomycin sulfate (HM). Each ovary was then punctured 10-20 times with a 27-gauge needle, incubated for 8 min in HM medium containing 6.8 mM EGTA, and centrifuged at 600 X g for 10 min, and the supernatant was discarded. This step was followed by 4 min incubation in HM medium containing 0.5 mM sucrose and 1.8 EDTA, and centrifugation at 600 x g for 10 min before the supernatant was discarded. The ovarian tissue was gently expressed through a stainless steel grid with a blunt microspatula in sterile HM without EGTA or sucrose to release all remaining granulosa cells. Clumps of tissue were removed by repeated decanting with a pipette, and cells were collected into the same tubes used for processing the ovaries to recover cells released during puncturing of ovaries. The supernatant was centrifuged again to sediment the cells, which were then resuspended in 40 ml HM medium. An aliquot of the cell suspension was counted in a hemocytometer; cell viability, assessed by trypan blue exclusion in protein-free medium, was 80-90%. Cell culture
Materials and Methods McCoy's 5a medium, penicillin-streptomycin solution, and L-glutamine were obtained from the NIH Media Unit (Bethesda, MD). DNase and PBS were purchased from Flow Laboratories (McLean, VA); BSA, EDTA, HEPES, polyethylene glycol 8000, trypan blue stain, and sucrose were purchased from JT Baker Company (Phillipsburg, NJ). All steroids were obtained from Steraloids (Wilton, NH). Pregnyl (hCG) was supplied by Organon Inc. (West Orange, NJ). Ovine (o)FSH was supplied by the National Hormone and Pituitary Program (Baltimore, MD). Tissue culture plasticware was from Falcon (Los Angeles, CA), Costar (Cambridge, MA) and/or Nalgene (Rochester, NY). Normal rabbit serum was obtained from GIBCO (Grand Island, NY). [His1,Nle27]hGRF(l-32)NH2, rGRF, VIP, PHI, secretin, gastric inhibitory peptide (GIP), glucagon, /3-endorphin, and CRF were purchased from Peninsula Laboratories (Belmont, CA) and Bachem (Torrance, CA) in order to make interbatch comparisons. [125I]VIP (2000 Ci/ mmol) was purchased from Amersham (Arlington Heights, IL) and [125I][His\Nle27]hGRF(l-32)NH2 (2200 Ci/mmol) was prepared by DuPont-New England Nuclear (Billerica, MA). Succinyl cAMP [125I]tyrosine methyl ester (2000 MCi/Mg),[125I]hCG ), [125I]histamine-CMO-progesterone,and [125I]estra-
Aliquots containing 2 X 105 viable granulosa cells were added to 24-well plastic culture plates in a total volume of 1 ml media without EGTA or sucrose. Various concentrations of oFSH (NIH-FSH-S13; FSH activity = 15 x NIH-FSH-Sl U/mg, LH activity = 0.05 x NIH-LH-Sl U/mg) and/or reagents, as specified in the text and legends, were added immediately before cell culture. Phosphodiesterase activity was inhibited by adding 0.4 mM MIX to the incubation medium. Androstenedione (10~7 M) was added as aromatase substrate in cultures used for studies on estradiol production. In some experiments, cells were primed with FSH for 72 h and washed four times with medium to remove accumulated endogenous hormones, and reagents were added for a time period up to 48 h; 4 x 105 cells were cultured in 12 X 75 mm polypropylene tubes. All cell cultures were maintained at 37 C in a humidified 95% air-5% CO2 environment for the specified incubation periods; the media were then removed and stored at —20 C for subsequent analysis of steroids and intracellular and extracellular cAMP production. RIAs Progesterone accumulation in the medium was determined by specific RIA on unextracted samples, using an antiserum raised against progesterone 11-BSA. Progesterone standards
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS and samples were incubated overnight at 4 C, and bound progesterone was separated from free by second antibody precipitation using 7.5% polyethylene glycol in Tris buffer. Estradiol was measured by a specific RIA without column chromatography. The cAMP RIA employed as the radiolabeled ligand 2' ,O-monosuccinyl-adenosine-3' ,5' -monophosphate tyrosine methyl ester, which was iodinated using the chloramine-T method. Labeled ScAMP-TME was purified by column chromatography on Sephadex-GlO using a water-methanol eluting buffer. The antiserum used was raised against the monosuccinyl cAMP-tyrosine methyl ester conjugated to BSA and did not react with other cyclic nucleotides. Sample media were heated at 100 C for 10 min immediately after removal from plates to abolish all phosphodiesterase activity. The cAMP standard and samples were routinely acetylated using triethylamine and acetic anhydride to optimize the sensitivity of the assay, which was 2 fmol/tube. Tubes were incubated in sodium acetate buffer containing 2.5% normal rabbit serum overnight, and the bound fraction was separated by the double antibody method. Binding studies LH receptors were measured by saturation binding assays with [125I]iodo-hCG, prepared by the chloramine-T method as previously described (43). After 72 h of culture, the polypropylene tubes containing the cell suspension were centrifuged at 600 X g for 15 min at room temperature. The cell pellets were washed twice with 1.5 ml aliquots of PBS, pH 7.4, containing with 0.1% BSA (PBS/BSA); 4-5 ng [125I]iodo-hCG (50,000100,000 cpm/ng) were added to tubes in a total volume of 300 fd; nonspecific binding was determined in the presence of an excess (100 IU) of unlabeled hCG. After incubation for 15-20 h at room temperature, cell-bound tracer was isolated by addition of 1.5 ml cold PBS/BSA and centrifugation at 1800 x g for 30 min. After aspiration of the supernatant, the step was repeated and bound radioactivity was measured by 7-spectrometry; nonspecific binding was subtracted to give specifically bound counts, which were converted to picograms of bound hCG per 2 X 105 cells. The bound hormone represents total LH receptor content since binding was measured with a saturating concentration of [125I]hCG. Binding assays with [125I]VIP and [^IHHisSNle^JhGRFU32)NH2 were performed on 3 X 106 granulosa cells suspended in 400 fi\ binding buffer (50 mM Tris-HCl, pH 7.4, containing 2 mM EGTA, 10 mM MgCl2) 5 mM CaCl2, 5 mM MnCl2, 60 ng/ ml bacitracin, and 0.1% BSA), with addition of 100 pM tracer in a final incubation volume of 500 /xl for selected time periods up to 0-120 min at different temperatures (4, 22, and 37 C). For equilibrium binding studies with [125I]VIP, cells were incubated at 37 C with increasing concentrations of the radioactive tracer in the absence or presence of an excess (10~6 M) of the corresponding unlabeled hormone. For binding-inhibition studies, cells were incubated with [125I]VIP or [125I][His\Nle27] hGRF(l-32)NH2 and several peptides of the glucagon family and unrelated peptides. The reaction was terminated by transferring 450 M1 incubation buffer into polyethylene microfuge tubes followed by centrifugation for 4 min. After aspiration of the supernatants, the tip of each tube containing the cell-bound
2119
tracer was severed, and the bound radioligand was quantified in a 7-spectometer. Statistical methods Each data point represents the mean ± SE of three to six replicates. Data were analyzed by Student's t test and analysis of variance. Scatchard analyses were performed using the LIGAND computer program. For binding assays, each point represents the mean of three experiments each performed in triplicate.
Results Specific binding of [125I]VIP to dispersed rat granulosa cells occurred rapidly and was time and temperature dependent. As shown in Fig. 1A, specific binding was maximal after 30 min incubation at 37 C and remained constant for 60 min. At 22 and 4 C the maximal specific binding was attained by 10 and 30 min, respectively, and was only 30% of that observed at 37 C; at these temperatures the maximum binding was followed by a decline, whereas at 37 C it was maintained for up to 60 min. Subsequent binding studies using [125I]VIP were performed at 37 C for 30 min. The properties of the granulosa cell receptors were also analyzed by binding studies 2000
Q- XI
> ; f >B
1000
il 60
90
Time (minutes)
Time (minutes)
FIG. 1. Kinetics of specific binding of [125I]VIP (A) and [125I] [His\Nle27]GRF(l-32)NH2 (B) to dispersed rat granulosa cells. Specific binding of each radioligand was measured for up to 120 min at temperatures of 4, 22, and 37 C. Nonspecific binding for each time interval was determined by the addition of the respective unlabeled peptide (1 jtM). Each point represents the mean of three determinations, with SE of less than 6%.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
2120
performed with [125I][His\Nle2]hGRF(l-32)NH27, a GRF analog with high biological activity (41). This radioligand exhibited rapid, time- and temperature-dependent specific binding to granulosa cells. Maximal binding was observed at 30 min incubation at 4 C and was maintained for up to 120 min (Fig. IB). At 22 and 37 C, maximum specific binding was observed by 20 and 30 min, respectively. At 37 C, maximal binding was followed by a rapid decline. Nonspecific binding, measured in the presence of 10~6 M unlabeled peptide, was less than 30% of total binding. Scatchard analysis of the saturation isotherm for [125I] VIP binding in granulosa cells incubated with increasing concentrations of radioligand (Fig. 2A) showed a high affinity binding site with dissociation constant (Kd) of
100
Endo • 1990 Vol 127 • No 5
A
• A D O
^ ^ •\ « GlucagonN
A GIP D CRF
11 m
CO
a. E
50
\
N^ N ^.
\ ^
\ *K
- O /3-endorphin
\^
\
Ni >v
\ ^
O PHI
^ N^
N-
•
Secretin
•
GRF (1-32)
\ ^ ^ ^ ^ * ^
A rGRF
• VIP I
^
n -w,
10"
I1
I
9
10"
10"
I 10'
i
1 i
i io-6
8
[Peptide] M
0.16 nM and binding capacity of 0.58 fmol per 106 cells,
equivalent to about 400 receptor sites per granulosa cell. Analysis of the inhibition of [125I] VIP binding by increasing concentrations of unlabeled peptide (Fig. 2B) revealed the presence of an additional low-affinity site with Kd of 15 nM and binding capacity of 87 fmol/106 cells. Evaluation of the ligand specificity of the granulosa cell binding sites (Fig. 3A) revealed half-maximum inhibitory concentrations (IC50) of 4.2 X 10"10 M for VIP, 9.3 x 10~9
B
100
#
A
D O
• Glucagon ^ ^ N ^
2 E
^
A GIP DCRF
\
O /3-endorphin
50
-
OPHI • Secretin
0.6
.A •
• GRF (1-32)
•
-
\
^
0
«s
\
• rGRF
8 0.4
v
• VIP
K 0 =1.6x10"'° Bma>-=0.S8lmol/t09ceDs Sites/Cell: 400
.,
I 10
I 1 0 -10
I
I 9
10"
^ i
8
10"
10"
:
1
i 6
10"
—A
>«.
1 10' 5
[Peptide] M
o
0.2
£
-f
,o
7 I
°
" A.
)
I
I
0.2 0.4 0.6 Bound (mol/106 cells 1
1
0.4
0.5
!
0.3
125
I-VIP nM
[VIP] M
FlG. 2. A, Saturation curve and Scatchard analysis of the high affinity VIP binding site in rat granulosa cells. Binding of increasing concentrations of [125I]VIP was measured at 37 C for 30 min. B, Bindinginhibition curve and Scatchard analysis of high and low affinity VIP binding sites. Cells were incubated with [125I]VIP in the presence of increasing concentrations of native VIP. Binding is expressed as a percentage of the [125I]VIP bound in the absence of unlabeled peptide.
FIG. 3. A, Inhibition of [125I]VIP binding by GRF and VIP-related peptides. Granulosa cells were incubated for 30 min at 37 C in the presence of 0.1 nM [125I]VIP and increasing concentrations of VIPrelated and other peptides. Binding is expressed as a percentage of the [125I]VIP bound in the absence of unlabeled peptide. B, Inhibition of [125I][His\Nle27]GRF(l-32)NH2 binding by GRF and VIP-related peptides. Cells were incubated for 45 min at 4 C in the presence of [125I] [His\Nle27]GRF(l-32)NH2 and increasing concentrations of rGRF(l43)OH, [His1,Nle27]GRF(l-32)NH2) VIP, secretin, PHI, GIP, glucagon, /3-endorphin, and CRF. Binding is expressed as a percentage of the [^IHHisSNle27] GRF(1-32)NH2 bound in the absence of unlabeled peptide. Each point represents the mean of three determinations with SE of less than 6%.
M for rGRF, 6.7 x 10~8 M for [His\Nle27]hGRF(l32)NH2, 8.5 X 10"8 M for PHI, and 5.2 x 10"7 M for secretin. GIP, glucagon, and the unrelated peptides, /?endorphin and CRF, did not inhibit [125I]VIP binding. The half-maximum inhibitory concentrations of the VIP-related peptides required to inhibit binding of the [125I]GRF analog differed from those observed with [125I] VIP as tracer, and the relative potencies were: rGRF(l43)OH > [His\Nle27]hGRF(l-32)NH2 > VIP > secretin > PHI (Fig. 3B). The IC50 of rGRF (1 x 10"9 M) was almost 2 orders of magnitude higher than that of VIP,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2121
GRF ACTIONS ON GRANULOSA CELLS
indicating the presence of a GRF-preferring receptor. The unrelated peptides, /3-endorphin and CRF, did not inhibit binding of the radioiodinated GRF analog. The effects of rGRF on cAMP and steroid production, and expression of LH receptors, were studied in basal and FSH-stimulated granulosa cells. GRF (10~7 M) alone stimulated cAMP production as shown by a modest increase in intracellular cAMP levels (Fig. 4A) and a larger rise in extracellular cAMP (Fig. 4B). However, in cells treated with FSH (100 ng/ml), GRF caused substantial elevations of intra- and extracellular cAMP at concentrations of 10~10 to 10"7 M, with ED50 of 10~9 M. The stimulation of cAMP production by GRF was accompanied by parallel enhancement of FSH-induced progesterone (Fig. 5A) and estradiol (Fig. 5B) production. The ability of GRF to amplify the FSH-induced acquisition of steroid biosynthetic capacity and aromatase activity was dose-dependent, with an ED50 of about 10~8 M. Basal progesterone and estradiol levels were unaffected by rGRF treatment.
Basal
FSH 50 ng
FSH 250 ng
FSH 1000 ng
B T 40 30-
T
o
*
^ 20 V> m
10 0
0
rGRF FSH [10-7]M 100 ng
1 0 -o
10
[rGRF] M
-7
1Q -6
Basal
FSH 50 ng
i
_ FSH 250 ng
J
FSH 1000 ng
FIG. 5. Enhancement of FSH-stimulated progesterone (A) and estradiol (B) production by GRF. Granulosa cells (4 x 106 cells/ml) were cultured for 72 h with increasing amounts of FSH in the absence (solid bars) or presence (open bars) of GRF (10~7 M). Each point represents the mean ± SE of triplicate determinations. *,P< 0.05; **, P < 0.01.
The stimulatory effects of GRF on FSH-induced granulosa cell responses were compared with those exerted by VIP. The dose-dependent increases in cAMP accumulation in response to both peptides are shown in Fig. 6A. In terms of the amplification of FSH-induced cAMP accumulation, VIP was more potent and more effective than GRF. The higher potency and efficacy of VIP were also evident in the enhancement of FSH-induced progesterone production (Fig. 6B) and aromatase activity (data LU not shown). The kinetics of the actions of 10~8 M VIP and rGRF on progesterone (Fig. 7A) and estradiol (Fig. v11 1 0 -10 1 0 -9 1Q -8 1Q -7 1Q -6 rGRF FSH 10" 7B) production were determined in cells treated for 72 h [10"7]M 100 ng [rGRF] M with 100 ng oFSH and cultured for a further 48 h in the presence of FSH and both peptides. Compared to conFIG. 4. Stimulation of intracellular (A) and extracellular (B) cAMP accumulation by GRF. Granulosa cells were cultured for 72 h in the trols (FSH alone), both VIP and rGRF increased steroid absence (solid bars) or presence of FSH (100 ng/ml) (hatched bars) and accumulation, and VIP was more effective than rGRF. n 6 increasing concentrations of rGRF (10" to 10" M); C, Control. Each The stimulatory effects of VIP and GRF on expression point represents the mean ± SE of triplicate determinations.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
• VIP
50
f
ArGRF
c ,o o
40 30
3
Endo • 1990 Vol 127 • No 5
80 - A
Pro
(ng/ ml)
2122
/
OFSH
/
/
/
/
/
/
I r
gest eron
-
f
o
//
Oin
J= ^ _L X
8«
300
-
/
XX /^-" /
200
"5 a. Q.
100 H
0
i
250 ng FSH
10" 11
i 10" 10
I 109
/
> r rGRP *
I 10"
I 8
10'
7
fPeptide] M
o
14
_£.
]B i
i
i
• 5 ^ 12 m o 10
al
2
CO
rt
a.
•
•
VIP
GRF
i
o E 4
•1 Control
GRF
VIP
1 FSH
GRF
VIP
GRF
•10 ng-
FIG. 8. Potentiation of FSH-induced LH receptor expression by GRF and VIP. A, Granulosa cells were cultured for 72 h in the presence of
FSH (250 ng/ml) and the indicated concentrations of GRF and VIP. At the end of the incubation, cells were assayed for [125I]hCG binding as described in Materials and Methods. Each point represents the mean ± SE of triplicate determinations. B, Effects of GRF and VIP on LH receptor expression. Granulosa cells were cultured for 72 h with 10~7 M GRF and/or VIP in the absence and presence of 10 ng FSH. Bars indicate the mean ± SE of triplicate determinations. *, P < 0.05.
brain (26), and liver (47, 48). However, only one class of binding sites is present in guinea pig intestinal cells (49) and human peripheral lymphocytes (50). The affinities of the two classes of VIP receptors observed in rat granulosa cells were similar to those reported in other VIP target tissues. There is reasonable agreement between the affinity of the VIP binding sites and the ED50 of VIP-induced responses in granulosa cells in the present and earlier studies (35, 36), as well as in the pituitary [where it stimulates PRL release (51)], in rat gut epithelium, and in the GH3 line of rat pituitary tumor cells (52). A major finding of this study was the potent agonist activity of rGRF(l-43)OH in terms of binding to the VIP receptor and stimulation of cAMP production, similar to its potency in guinea pig pancreas (53). Specific high affinity binding sites for GRF have been described only
2123
in anterior pituitary cells (41, 54, 55) and pituitary membrane homogenates (56), where VIP and GRF receptors are distinct entities with different cellular locations (lactotropes vs. somatotropes) and mechanisms of action, and stringent selectivities for the two peptides. In terms of cAMP production, VIP is a weak agonist at the pituitary level (57) but is a more potent stimulus in the granulosa cell. Studies on the GRF receptor have been facilitated by the use of the potent analog, [125I] [His1,Nle27]hGRF(l-32)NH2, as tracer in the binding assay (41). In rat granulosa cells, the optimal conditions for binding of the 125I-labeled GRF agonist differed markedly from those of [125I]VIP, with maximal specific binding at 4 C rather than 37 C. The level of specific binding of the GRF radioligand was less than that of [125I]VIP, and the relative binding-inhibition potencies for VIP and the GRF peptides were reversed, with rGRF(l-43)OH > [His1,Nle27]GRF(l-32)NH2 > VIP > PHI > secretin. The finding that GRF is almost 1 order of magnitude more potent than VIP in competing with the GRF radioligand for receptor binding suggests that the common receptor for GRF and VIP adopts a GRF-preferring confirmation at low temperature. However, GRF is a relatively less potent agonist than VIP in eliciting biological responses of granulosa cells incubated at 37 C, a difference reflected in its lower activity in [125I] VIP binding-inhibition studies at this temperature. It is likely that the peptides of the VIP/GRF family have evolved through successive events of gene duplication from a single ancestral gene coding for an original peptide (47), with mutations to acquire the structural properties necessary to interact with different sets of receptors. This evolutionary process could be accompanied by changes not only in the peptides, but also in the membrane proteins forming the receptor site. At the ovarian level, as in other target tissues, it is possible that a family of receptors for peptides structurally related to VIP has evolved in parallel with the peptides themselves. The potential functional role of such receptors in the ovary is indicated by the known actions of VIP on granulosa cell development (35-39) and the presence of VIP in the gonads, as noted earlier. It is likely that GRF is also formed in the gonads and acts locally on the VIP receptor. GRF-like material has been detected by RIA in extracts of rat ovary (Moretti C. and A. Bagnato, unpublished data) and in human follicular fluid (16), and by immunostaining in the interstitium of the human ovary (15). Also, a GRF-like factor and its mRNA have been identified in the rat testis (14). GRF itself had only minor effects on granulosa cell function, but potentiated FSH-induced granulosa cell differentiation in a dosedependent manner. The concentrations of GRF required to amplify the stimulatory effects of FSH on granulosa cell activity were consistent with its relative binding
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2124
GRF ACTIONS ON GRANULOSA CELLS
affinity at the VIP/GRF receptor in these cells. It is possible that the presence of adequate concentrations of locally synthethized GRF in the interstitial fluid could modulate granulosa cell responsiveness to gonadotropic stimulation. In infertile patients resistant to gonadotropin therapy, cotreatment with the bioactive N-terminal portion of the peptide (GRF 1-29) enhances the stimulatory action of gonadotropins on ovarian function (16). This effect of exogenous GRF on the human ovary could be exerted through at least two distinct mechanisms. First, the increased production of GH and somatomedin-C (SmC)/ insulin-like growth factor I (IGF-I) would be expected to increase the ovarian response to gonadotropin stimulation. Rat granulosa cells are responsive to GH (58, 59) and SmC/IGF-l (60), both of which potentiate the actions of FSH on differentiation and steroidogenesis. GH does not appear to stimulate the expression of mRNA for SmC/IGF-I or IGF-II in human ovarian tissue (61), but specific receptors for SmC/IGF-I have been recently identified in human granulosa cells (62). Second, the sensitizing action of GRF 1-29 could also be due to a direct action of the peptide on granulosa-cell receptors for peptides of the VIP family, leading to amplification of the actions of FSH on follicle growth and steroidogenic capacity. In the present studies, rGRF caused marked increases in FSH-stimulated cAMP and progestin production, aromatase activity, and expression of LH receptors. These effects were not additive to those of VIP, which caused higher maximal increases in most responses and was about 1 order of magnitude more potent than rGRF. The finding that GRF, as well as VIP, enhances all aspects of granulosa cell differentiation suggests that these peptides can modulate the trophic action of FSH in the control of follicular maturation. The ability of GRF and VIP to enhance FSH-stimulated cAMP production is probably the primary mechanism through which the peptides potentiate granulosa cell differentiation. Although the efficacy of GRF per se is relatively low, its ability to stimulate cAMP production is consistent with the actions of GRF in pituitary somatotrophs (1, 2) and in those peripheral tissues in which GRF recognizes the VIP-receptor, namely human and rat intestinal epithelial cells (63), and rat pancreatic liver membranes (47). The present findings add rGRF to the several peptides known to stimulate the adenylate cyclase-cAMP system in ovarian granulosa cells (64-66). The nature of the dose-response curves for cAMP production and biological actions in granulosa cells indicates that small degrees of VIP/GRF receptor occupancy significantly amplify the FSH-induced adenylate cyclase response. Thus, if FSH is present in low concentrations, the synergistic action of GRF could significantly enhance granulosa-cell responsiveness and accelerate the matu-
Endo • 1990 Vol 127 • No 5
ration process. In conclusion, the receptor-mediated enhancement by GRF of FSH-induced steroidogenesis and LH/hCG receptor expression is probably exerted through the interaction of the peptide with VIP/GRF receptors present on the surface of granulosa cells. The different incubation conditions required for optimal binding of VIP and the GRF-agonist, and the reciprocal affinities of the two structurally related peptides in homologous radioligand binding studies, suggest that the selectivity of the receptor for VIP and GRF is temperature dependent. These findings, and the localization of GRF in ovarian interstitial cells (15), indicate that GRF can exert paracrine regulation, similar to the action of VIP, on ovarian function. In this manner, locally produced or exogenously administered peptide would exert direct positive modulation of trophic hormone-stimulated maturation of the granulosa cell. Since it lacks the hypotensive action of VIP, GRF could provide a therapeutic measure for the activation of ovarian VIP/GRF receptors when local potentiation of gonadotropin action is required for optimal stimulation of ovarian follicular development. Acknowledgments The authors are grateful to Dr. S. Ulisse for helpful discussions and technical advice. This work was supported in part by a grant from Industria Pharmaceutica Serono, Rome, Italy.
References 1. Frohman LA, Jansson GO 1986 Growth hormone releasing hormone. Endocr Rev 7:223 2. Spiess J, Rivier J, Vale W 1983 Characterization of rat hypothalamic growth hormone-releasing factor. Nature 303:532 3. Shibasaki T, Kiyosawa Y, Masuda A, Nakahara M, Imaki T, Wakabayashi I, Demura H, Shizume K, Ling N 1985 Distribution of growth hormone-releasing-factor-like immunoreactivity in human tissues. J Clin Endocrinol Metab 59:263 4. Thorner MO, Vance ML, Evans WS, Ho K, Rogol AD, Blizzard RM, Furlanetto R, Rivier J, Vale W 1986 Growth hormone releasing factor and somatomedin C production: extrahypothalamic localization and possible functional significance. Acta Endocrinol [Suppl] (Copenh) 276:34 5. Ling N, Zeytin F, Bohlen P, Esch F, Brazeau P, Wehrenberg WB, Baird A, Guillemin R 1985 Growth hormone releasing factors. Annu Rev Biochem 54:403 6. Christofides ND, Stephanou A, Suzuki H, Yiangou Y, Bloom SR 1984 Distribution of immunoreactive growth hormone releasing hormone in the human brain and intestine and its production by tumors. J Clin Endocrinol Metab 59:747 7. Bruhn TO, Mason RT, Vale W 1985 Presence of growth-hormonereleasing-factor-like immunoreactivity in rat duodenum. Endocrinology 117:1710 8. Losa M, Wolfram G, Mojto J, Schopohl J, Spiess Y, Huber R, Muller OA, von Werder K 1990 Presence of growth hormonereleasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties. J Clin Endocrinol Metab 70:62 9. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276 10. Nicholson WE, DeCerney GS, Jackson RV, Orth DN 1987 Pitui-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
11. 12. 13.
14. 15.
16.
17.
18. 19. 20.
21.
22. 23.
24.
25.
26.
27. 28.
tary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin. Regul Peptides 18:173 Baird A, Wehrenberg WB, Bohlen P, Ling N 1985 Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117:1598 Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098 Margioris NM, Brockmann G, Bohler Jr HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 126:151 Berry SA, Pescovitz OH 1988 Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 123:661 Moretti C, Fabbri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazzicone Q, Spera G 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads. J Endocrinol Invest 13:301 Moretti C, Fabbri A, Gnessi L, Forni L, Fraioli F, Frajese G 1989 GHRH stimulates follicular growth and amplified FSH-induced ovarian folliculogenesis in women with anovulatory infertility. In: Frajese G, Steinberg E, Rodriguez-Rigau LJ (eds) Reproductive Medicine: Medical Therapy. Proceedings of the Second International Symposium on Reproductive Medicine. Elsevier Science Publishers, New York, no 875, pp 103-112 Christophe J, Svoboda M, Dehaye JP, Winand J, VandermeersPire MC, Vandermeers A, Cauvin A, Gourlet P, Robberecht P 1989 The VIP/PHI/secretin/helodermin/helospectin/GRF family: structure-function relationship of the natural peptides, their precursors and synthetic analogues as tested in vitro on receptors and adenylate cyclase in a panel of tissue membranes. In: Martinez J (ed) Peptide Hormones as Prohormones: Processing, Biological Activity, Pharmacology. Halsted Press, New York, pp 211-242 Bataille D 1986 VIP and related substances. Peptides 7 [Suppl 1] 1:390 Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides 7 [Suppl 1] 1:27 Schwartz CJ, Kimberg DV, Sheerin HE, Field M, Said SI 1974 Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54:536 Feliu JE, Mojena M, Silvestre RA, Monge L, Marco J 1983 Stimulatory effect of VIP on glycogenolysis and gluconeogenesis in isolated rat hepatocytes: antagonism by insulin. Endocrinology 112:2120 Said SI, Mutt V 1970 Polypeptide with broad biological activity: isolation from small intestine. Science 169:1217 Bataille D, Freychet P, Rosselin G 1974 Interactions of glucagon, gut glucagon, vasoactive intestinal polypeptide and secretin with liver and fat cell plasma membranes: binding to specific sites and stimulation of adenylate cyclase. Endocrinology 95:713 Bataille D, Besson J, Bastard C, Laburthe M, Rosselin G 1977 Specificity in hormone-receptor interaction: studies with insulin, glucagon and vasoactive intestinal peptide (VIP). In: Bonfils S, Fromageot P, Rosselin G (eds) Hormonal Receptors in Digestive Tract Physiology. North Holland, Amsterdam, p 113-119 Dharmsathaphorn K, Harms V, Binder HJ, Yamashiro DJ, Wright EM 1983 Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J Clin Invest 75:462 Staun-Olsen P, Ottesen B, Bartels D, Nielsen MH, Gammeltoft S, Fahrenkrug J 1982 Receptors for vasoactive intestinal polypeptide on isolated synaptosomes from rat cerebral cortex. Heterogeneity of binding and desensitization of receptors. J Neurochem 39:1242 Christophe J, Conlon TP, Gardner JD 1976 Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. J Biol Chem 251:4629 Robberecht P, Waelbroeck M, Camus JC, DeNeef P, Christophe J 1984 Importance of disulfide bonds in receptors for vasoactive
29. 30.
31. 32. 33. 34.
35. 36.
37.
38.
39.
40. 41. 42. 43. 44.
45. 46. 47. 48.
49.
2125
intestinal peptide and secretin in rat pancreatic plasma membranes. Biochim Biophys Acta 773:271 Prieto JC, Carmena MJ 1983 Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate. Biochim Biophys Acta 763:408 Zhen-Guo L, Queen G, LaBella FS 1990 Adrenocorticotropin, vasoactive intestinal polypeptide, growth hormone-releasing factor, and dynorphin compete for common receptors in brain and adrenal. Endocrinology 126:1327 Wanke IE, Rorstad OP 1990 Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to lactotropes. Endocrinology 126:1981 Larson LI, Fahrenkrug J, Schaffalitzky de Muckadell OB 1977 Vasoactive intestinal polypeptide occurs in nerves of the female genitourinary tract. Science 197:1374 Aim P, Alumets J, Hakanson R, Helm G, Owman C, Sjoberg NO, Sundeler F 1980 Vasoactive intestinal polypeptide nerves in the human female genital tract. Am J Obstet Gynecol 136:349 Klein CM, Burden HW 1988 Substance P and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers in relation to ovarian postganglionic perikarya in para- and prevertebral ganglia: evidence from combined retrograde tracing and immunocytochemistry. Cell Tissue Res 252:403 Davoren BJ, Hsueh AJW 1985 Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. Biol Reprod 33:37 Trzeciak WH, Ahmed CE, Simpson ER, Ojeda SR 1986 Vasoactive intestinal peptide induces the synthesis of the cholesterol sidechain cleavage enzyme complex in cultured rat ovarian granulosa cells. Proc Natl Acad Sci USA 83:7490 George FW, Ojeda SR 1987 Vasoactive intestinal peptide enhances aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone. Proc Natl Acad Sci USA 84:5803 Tornell J, Carlsson B, Hillensjo T 1988 Vasoactive intestinal peptide stimulates oocyte maturation, steroidogenesis and cyclic adenosine 3',5' monophosphate production in isolated preovulatory rat follicles. Biol Reprod 39:213 Liu YX, Kasson BG, Dahl KD, Hsueh AJW 1987 Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes. Peptides 8:29 Gozes I, Tsafriri A 1986 Detection of vasoactive intestinal peptideencoding messenger ribonucleic acid in the rat ovaries. Endocrinology 119:2606 Seifert H, Perrin M, Rivier J, Vale W 1985 Binding sites for growth hormone releasing factor on rat anterior pituitary cells. Nature 313:487 Knecht M, Katz MS, Catt KJ 1981 Gonatropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells. J Biol Chem 256:34 Catt KJ, Dufau ML 1975 Gonadal receptors for LH and chorionic gonadotropin. Methods Enzymol 37:167 Christophe J, Svoboda M, Lambert M, Waelbroeck M, Winand J, Dehaye JP, Vandermeers-Piret MC, Vandermeers A, Robberecht P 1986 Effector mechanism of peptides of the VIP Family. Peptides 7 [Suppl l]:101 Laburthe M, Couvineau A 1988 Molecular analysis of vasoactive intestinal peptide receptors. A comparison with receptors for VIPrelated peptides. Ann NY Acad Sci 527:296 Laburthe M, Breant B, Rouyer-Fessard C 1984 Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Eur J Biochem 139:181 Christophe J, Svoboda M, Magali W, Winand J, Robberecht P 1988 Vasoactive intestinal peptide receptors in pancreas and liver. Ann NY Acad Sci 527:238 Couvineau AB, Amiranoff B, Laburthe M 1986 Solubilization of the liver VIP receptor: hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein. J Biol Chem 261:8527 Binder HJ, Lemp GF, Gardner JP 1980 Receptors for vasoactive intestinal peptide and secretin in small intestinal epithelial cells.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2126
GRF ACTIONS ON GRANULOSA CELLS
Am J Physiol 238:190 50. Danek A, O'Dorisio MS, O'Dorisio TM, George JM 1983 Specific binding sites for vasoactive intestinal polypeptide on nonadherent peripheral blood lymphocytes. J Immunol 131:1173 51. Rotsztejn WH, Dussaillant M, Nobou F, Rosselin G 1981 Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture. Proc Natl Acad Sci USA 78:7584 52. Zeytin FN, Reyl-Desmars F, Rathbun T 1985 Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP-prefering receptor site(s). Biochem Biophys Res Comraun 127:992 53. Pandol SJ, Seifert H, Thomas MW, Rivier J, Vale W 1984 Growth hormone-releasing factor stimulates pancreatic enzyme secretion. Science 225:326 54. Valicelebi G, Santacroce TM, Harpold MM 1985 Specific binding of synthetic human pancreatic growth hormone releasing factor (140 OH) to bovine anterior pituitaries. Biochem Biophys Res Commun 126:33 55. Ikuyama S, Natori S, Nawata H, Kato KI, Ibayashi H, Kariya T, Sakai T, Rivier J, Vale W 1988 Characterization of growth hormone-releasing hormone receptors in pituitary adenomas from patients with acromegaly. J Clin Endocrinol Metab 66:1265 56. Seifert H, Perrin M, Rivier J, Vale W 1985 Growth hormonereleasing factor binding sites in rat anterior pituitary membrane homogenate: modulation by glucocorticoids. Endocrinology 117:424 57. Magistretti PJ, Schoenberg P, Kehrer P, Martin JL, Gaillard RC 1986 Actions of VIP, hGRF, PHI and Secretin: comparative studies in cerebral cortex and adenohypophysis. Peptides 7[Suppl 1]:175 58. Jia XC, Kalmijn J, Hsueh AJW 1986 Growth hormone enhances
59. 60.
61.
62. 63.
64.
65. 66.
Endo • 1990 Vol 127 • No 5
follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 118:1601 Davoren JB, Hsueh AJW 1986 Growth hormone increases ovarian levels of immunoreactive somatomedin C/insulin-like growth factor I in vivo. Endocrinology 118:888 Adashi EY, Resnick CE, Brodie AMH, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells. Endocrinology 117:2313 Voultilainen R, Miller WL 1987 Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side chain cleavage enzyme, P450scc, in human steroidogenic tissue. Proc Natl Acad Sci USA 84:1590 Poretsky L, Kalin MF 1987 The gonadotropic function of insulin. Endocr Rev 8:132 Laburthe M, Couvineau A, Rouyer-Fessard C 1986 Study of species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal peptide (VIP) receptors using GRF and intestinal VIP receptors from rat and human: evidence that AcTyr1 hGRF is a competitive VIP antagonist in the rat. Mol Pharmacol 29:23 Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1986 Somatomedin-C as an amplifier of follicle-stimulating hormone action: enhanced accumulation of adenosine 3',5'-mono phosphate. Endocrinology 118:149 Knecht M, Catt KJ 1983 Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 258:2789 Knecht M, Feng P, Catt KJ 1989 Transforming growth factorbeta: autocrine, paracrine, and endocrine effects in ovarian cells. Semin Reprod Endocrinol 1:12
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
Vol. 127, No. 5 Printed in U.S.A.
Receptor-Mediated Actions of Growth Hormone Releasing Factor on Granulosa Cell Differentiation C. MORETTI, A. BAGNATO, N. SOLAN, G. FRAJESE, AND K. J. CATT Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD; and Clinica Medica V (G.F.), University La Sapienza, Rome, Italy
Glucagon and gastric inhibitory peptide, other peptides of the glucagon superfamily, and unrelated peptides including CRF and /3-endorphin, did not inhibit binding of either radioligand to ovarian receptors. In cultured granulosa cells, rGRF and VIP stimulated cAMP formation, consistent with coupling of their receptors to the adenylate cyclase system, and potentiated FSHinduced cAMP production. Both peptides also amplified FSHinduced progesterone biosynthesis, aromatase activity, and LH receptor formation. These observations demonstrate that rGRF is a potent cAMP-mediated agonist in the rat ovary and acts on a common VIP/GRF receptor in maturing granulosa cells. It is likely that the potentiating effect of administered GRF on gonadotropin-stimulated follicular development in vivo is in part mediated by direct actions of the peptide on the VIP/GRF receptor. Also, since GRF is present in the gonads, it is possible that the locally-produced peptide promotes follicular maturation by paracrine modulation of the stimulatory action of FSH on granulosa cell function. (Endocrinology 127: 2117-2126, 1990)
ABSTRACT. GRF promotes follicular maturation and ovulation when administered with FSH in the treatment of infertility. Such actions could be mediated by stimulation of GH secretion and insulin-like growth factor I production, but the known actions of the structurally related hormone, vasoactive intestinal peptide (VIP), on granulosa cell function suggested that GRF may also act directly on the ovary to stimulate follicular development. Radioligand binding and activation studies, performed in granulosa cells from immature estrogen-treated rats, revealed a common receptor for VIP and rat (r) GRF in the ovary. Specific binding of [125I]VIP to granulosa cells was saturable and dependent on time and temperature. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP > rGRF > peptide histidine isoleucinamide > [His\Nle27] human GRF(1-32)NH2 > secretin. In binding studies with the potent GRF agonist, [125I][His1,Nle27]GRF(l-32)NH2) relative potencies were: rGRF(l-43)OH > [HisSNle^Jhuman GRF(132)NH2 > VIP > peptide histidine isoleucinamide > secretin.
T
HE 43 amino acid hypothalamic polypeptide, GRF, is the major physiological stimulus of GH secretion from the anterior pituitary gland (1). In the rat, GRF was isolated from hypothalamic extracts on the basis of its ability to stimulate GH secretion from cultured anterior pituitary cells (2). GRF-like immunoreactivity has since been found in several extrahypothalamic sites (35), including the gastrointestinal tract (6, 7), human tumors (8, 9), tumors of neuroendocrine origin (10), and placental extracts (11-13), suggesting that GRF may also have a regulatory role in several peripheral tissues. At the gonadal level, a GRF-like substance and its messenger RNA (mRNA) have been identified in the postpuberal rat testis (14). In man, GRF-like material has been demonstrated in both ovarian and in testicular tissue by the immunoperoxidase technique (15), and immunoreactive GRF has been detected in ovarian follicular fluid Received July 18, 1990. Address requests for reprints to: Dr. Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 10, Room B1-L400, Bethesda, Maryland 20892.
(16). In infertile women resistant to gonadotropin therapy, the administration of GRF(l-29) in association with pure FSH activates folliculogenesis and stimulates the development of a dominant ovarian follicle (16). The amino acid sequence of rat (r) GRF has striking homologies with the sequence of the vasoactive intestinal peptide (VIP) peptide histidine isoleucinamide (PHI)/ secretin/Helodermin/Helospectin family of peptides (17, 18). Complementary DNA clones which encode the precursor to VIP and GRF have been isolated and, despite the distinction in size and sequence of the prepropeptides, the structural organization of the two precursors is similar (19). The homology between these two peptides also extends to their ability to elicit biological responses in several target tissues. The broad range of actions of VIP/PHI in various target cells includes alterations in water and electrolyte transfer in epithelial cells (20), stimulation of glycogenolysis in hepatocytes (21), and relaxation of smooth muscle (22). Specific recognition sites for VIP are present in rat liver (23) fat (24), intestine (25), brain (26), lung (27), pancreas (28), and prostate (29). In the brain and the adrenal gland, ACTH,
2117
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2118
GRF ACTIONS ON GRANULOSA CELLS
VIP, GRF, and dynorphin compete for common receptors (30). In the rat pituitary gland, receptors for VIP have been characterized on lactotropes (31). There is considerable evidence for a role of VIP in the control of reproductive function. Immunoreactive VIP and VIPergic nerve fibers are present in the female genital tract of animals (32) and humans (33), in association with the smooth muscle of blood vessels and in the ovarian stroma (34). VIP stimulates steroidogenesis in cultured rat granulosa cells (35), regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex which catalyzes the rate-limiting reaction in progesterone biosynthesis (36), and enhances aromatase activity in the neonatal rat ovary before the development of primary follicles, at a stage in which the follicles are not yet responsive to FSH (37). VIP also stimulates oocyte maturation and cAMP production in isolated preovulatory rat follicles (38), and plasminogen activator activity in avian granulosa cells (39). VIP-encoding mRNA has been detected in rat ovaries, suggesting local synthesis of the peptide (40). The present study was performed to analyze the interaction between GRF and the VIP receptor in rat granulosa cells. To this end, we characterized the specific binding of [125I]VIP and [125I][His\Nle27] human (h) GRF(1-32)NH2, a potent analog of rGRF (41), to these cells and compared the actions of these structurally related peptides on gonadotropin-induced differentiation and steroidogenesis in rat granulosa cells. These studies have revealed potent synergistic actions of both rGRF and VIP on FSH-induced granulosa cell maturation.
Endo • 1990 Vol 127 • No 5
diol were supplied by Hazleton Biotechnologies (Vienna, VA). 3-Isobutyl-l-methylxanthine (MIX) was purchased from Aidrich (Milwaukee, WI). Preparation of granulosa cells
Granulosa cells were prepared from ovaries of estrogentreated immature female rats as previously described (42). Silastic capsules (10 mm) containing diethylstilbestrol were implanted sc on day 21 to stimulate granulosa cell proliferation, and animals were killed between the ages of 25-32 days by decapitation. Ovaries were removed under aseptic conditions and dissected free of adherent connective tissue in sterile modified McCoy's 5a medium supplemented with 10 mM HEPES, pH 7.4, 4 mM L-glutamine, 100 U/ml penicillin, and 100 iig/rcA streptomycin sulfate (HM). Each ovary was then punctured 10-20 times with a 27-gauge needle, incubated for 8 min in HM medium containing 6.8 mM EGTA, and centrifuged at 600 X g for 10 min, and the supernatant was discarded. This step was followed by 4 min incubation in HM medium containing 0.5 mM sucrose and 1.8 EDTA, and centrifugation at 600 x g for 10 min before the supernatant was discarded. The ovarian tissue was gently expressed through a stainless steel grid with a blunt microspatula in sterile HM without EGTA or sucrose to release all remaining granulosa cells. Clumps of tissue were removed by repeated decanting with a pipette, and cells were collected into the same tubes used for processing the ovaries to recover cells released during puncturing of ovaries. The supernatant was centrifuged again to sediment the cells, which were then resuspended in 40 ml HM medium. An aliquot of the cell suspension was counted in a hemocytometer; cell viability, assessed by trypan blue exclusion in protein-free medium, was 80-90%. Cell culture
Materials and Methods McCoy's 5a medium, penicillin-streptomycin solution, and L-glutamine were obtained from the NIH Media Unit (Bethesda, MD). DNase and PBS were purchased from Flow Laboratories (McLean, VA); BSA, EDTA, HEPES, polyethylene glycol 8000, trypan blue stain, and sucrose were purchased from JT Baker Company (Phillipsburg, NJ). All steroids were obtained from Steraloids (Wilton, NH). Pregnyl (hCG) was supplied by Organon Inc. (West Orange, NJ). Ovine (o)FSH was supplied by the National Hormone and Pituitary Program (Baltimore, MD). Tissue culture plasticware was from Falcon (Los Angeles, CA), Costar (Cambridge, MA) and/or Nalgene (Rochester, NY). Normal rabbit serum was obtained from GIBCO (Grand Island, NY). [His1,Nle27]hGRF(l-32)NH2, rGRF, VIP, PHI, secretin, gastric inhibitory peptide (GIP), glucagon, /3-endorphin, and CRF were purchased from Peninsula Laboratories (Belmont, CA) and Bachem (Torrance, CA) in order to make interbatch comparisons. [125I]VIP (2000 Ci/ mmol) was purchased from Amersham (Arlington Heights, IL) and [125I][His\Nle27]hGRF(l-32)NH2 (2200 Ci/mmol) was prepared by DuPont-New England Nuclear (Billerica, MA). Succinyl cAMP [125I]tyrosine methyl ester (2000 MCi/Mg),[125I]hCG ), [125I]histamine-CMO-progesterone,and [125I]estra-
Aliquots containing 2 X 105 viable granulosa cells were added to 24-well plastic culture plates in a total volume of 1 ml media without EGTA or sucrose. Various concentrations of oFSH (NIH-FSH-S13; FSH activity = 15 x NIH-FSH-Sl U/mg, LH activity = 0.05 x NIH-LH-Sl U/mg) and/or reagents, as specified in the text and legends, were added immediately before cell culture. Phosphodiesterase activity was inhibited by adding 0.4 mM MIX to the incubation medium. Androstenedione (10~7 M) was added as aromatase substrate in cultures used for studies on estradiol production. In some experiments, cells were primed with FSH for 72 h and washed four times with medium to remove accumulated endogenous hormones, and reagents were added for a time period up to 48 h; 4 x 105 cells were cultured in 12 X 75 mm polypropylene tubes. All cell cultures were maintained at 37 C in a humidified 95% air-5% CO2 environment for the specified incubation periods; the media were then removed and stored at —20 C for subsequent analysis of steroids and intracellular and extracellular cAMP production. RIAs Progesterone accumulation in the medium was determined by specific RIA on unextracted samples, using an antiserum raised against progesterone 11-BSA. Progesterone standards
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS and samples were incubated overnight at 4 C, and bound progesterone was separated from free by second antibody precipitation using 7.5% polyethylene glycol in Tris buffer. Estradiol was measured by a specific RIA without column chromatography. The cAMP RIA employed as the radiolabeled ligand 2' ,O-monosuccinyl-adenosine-3' ,5' -monophosphate tyrosine methyl ester, which was iodinated using the chloramine-T method. Labeled ScAMP-TME was purified by column chromatography on Sephadex-GlO using a water-methanol eluting buffer. The antiserum used was raised against the monosuccinyl cAMP-tyrosine methyl ester conjugated to BSA and did not react with other cyclic nucleotides. Sample media were heated at 100 C for 10 min immediately after removal from plates to abolish all phosphodiesterase activity. The cAMP standard and samples were routinely acetylated using triethylamine and acetic anhydride to optimize the sensitivity of the assay, which was 2 fmol/tube. Tubes were incubated in sodium acetate buffer containing 2.5% normal rabbit serum overnight, and the bound fraction was separated by the double antibody method. Binding studies LH receptors were measured by saturation binding assays with [125I]iodo-hCG, prepared by the chloramine-T method as previously described (43). After 72 h of culture, the polypropylene tubes containing the cell suspension were centrifuged at 600 X g for 15 min at room temperature. The cell pellets were washed twice with 1.5 ml aliquots of PBS, pH 7.4, containing with 0.1% BSA (PBS/BSA); 4-5 ng [125I]iodo-hCG (50,000100,000 cpm/ng) were added to tubes in a total volume of 300 fd; nonspecific binding was determined in the presence of an excess (100 IU) of unlabeled hCG. After incubation for 15-20 h at room temperature, cell-bound tracer was isolated by addition of 1.5 ml cold PBS/BSA and centrifugation at 1800 x g for 30 min. After aspiration of the supernatant, the step was repeated and bound radioactivity was measured by 7-spectrometry; nonspecific binding was subtracted to give specifically bound counts, which were converted to picograms of bound hCG per 2 X 105 cells. The bound hormone represents total LH receptor content since binding was measured with a saturating concentration of [125I]hCG. Binding assays with [125I]VIP and [^IHHisSNle^JhGRFU32)NH2 were performed on 3 X 106 granulosa cells suspended in 400 fi\ binding buffer (50 mM Tris-HCl, pH 7.4, containing 2 mM EGTA, 10 mM MgCl2) 5 mM CaCl2, 5 mM MnCl2, 60 ng/ ml bacitracin, and 0.1% BSA), with addition of 100 pM tracer in a final incubation volume of 500 /xl for selected time periods up to 0-120 min at different temperatures (4, 22, and 37 C). For equilibrium binding studies with [125I]VIP, cells were incubated at 37 C with increasing concentrations of the radioactive tracer in the absence or presence of an excess (10~6 M) of the corresponding unlabeled hormone. For binding-inhibition studies, cells were incubated with [125I]VIP or [125I][His\Nle27] hGRF(l-32)NH2 and several peptides of the glucagon family and unrelated peptides. The reaction was terminated by transferring 450 M1 incubation buffer into polyethylene microfuge tubes followed by centrifugation for 4 min. After aspiration of the supernatants, the tip of each tube containing the cell-bound
2119
tracer was severed, and the bound radioligand was quantified in a 7-spectometer. Statistical methods Each data point represents the mean ± SE of three to six replicates. Data were analyzed by Student's t test and analysis of variance. Scatchard analyses were performed using the LIGAND computer program. For binding assays, each point represents the mean of three experiments each performed in triplicate.
Results Specific binding of [125I]VIP to dispersed rat granulosa cells occurred rapidly and was time and temperature dependent. As shown in Fig. 1A, specific binding was maximal after 30 min incubation at 37 C and remained constant for 60 min. At 22 and 4 C the maximal specific binding was attained by 10 and 30 min, respectively, and was only 30% of that observed at 37 C; at these temperatures the maximum binding was followed by a decline, whereas at 37 C it was maintained for up to 60 min. Subsequent binding studies using [125I]VIP were performed at 37 C for 30 min. The properties of the granulosa cell receptors were also analyzed by binding studies 2000
Q- XI
> ; f >B
1000
il 60
90
Time (minutes)
Time (minutes)
FIG. 1. Kinetics of specific binding of [125I]VIP (A) and [125I] [His\Nle27]GRF(l-32)NH2 (B) to dispersed rat granulosa cells. Specific binding of each radioligand was measured for up to 120 min at temperatures of 4, 22, and 37 C. Nonspecific binding for each time interval was determined by the addition of the respective unlabeled peptide (1 jtM). Each point represents the mean of three determinations, with SE of less than 6%.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
2120
performed with [125I][His\Nle2]hGRF(l-32)NH27, a GRF analog with high biological activity (41). This radioligand exhibited rapid, time- and temperature-dependent specific binding to granulosa cells. Maximal binding was observed at 30 min incubation at 4 C and was maintained for up to 120 min (Fig. IB). At 22 and 37 C, maximum specific binding was observed by 20 and 30 min, respectively. At 37 C, maximal binding was followed by a rapid decline. Nonspecific binding, measured in the presence of 10~6 M unlabeled peptide, was less than 30% of total binding. Scatchard analysis of the saturation isotherm for [125I] VIP binding in granulosa cells incubated with increasing concentrations of radioligand (Fig. 2A) showed a high affinity binding site with dissociation constant (Kd) of
100
Endo • 1990 Vol 127 • No 5
A
• A D O
^ ^ •\ « GlucagonN
A GIP D CRF
11 m
CO
a. E
50
\
N^ N ^.
\ ^
\ *K
- O /3-endorphin
\^
\
Ni >v
\ ^
O PHI
^ N^
N-
•
Secretin
•
GRF (1-32)
\ ^ ^ ^ ^ * ^
A rGRF
• VIP I
^
n -w,
10"
I1
I
9
10"
10"
I 10'
i
1 i
i io-6
8
[Peptide] M
0.16 nM and binding capacity of 0.58 fmol per 106 cells,
equivalent to about 400 receptor sites per granulosa cell. Analysis of the inhibition of [125I] VIP binding by increasing concentrations of unlabeled peptide (Fig. 2B) revealed the presence of an additional low-affinity site with Kd of 15 nM and binding capacity of 87 fmol/106 cells. Evaluation of the ligand specificity of the granulosa cell binding sites (Fig. 3A) revealed half-maximum inhibitory concentrations (IC50) of 4.2 X 10"10 M for VIP, 9.3 x 10~9
B
100
#
A
D O
• Glucagon ^ ^ N ^
2 E
^
A GIP DCRF
\
O /3-endorphin
50
-
OPHI • Secretin
0.6
.A •
• GRF (1-32)
•
-
\
^
0
«s
\
• rGRF
8 0.4
v
• VIP
K 0 =1.6x10"'° Bma>-=0.S8lmol/t09ceDs Sites/Cell: 400
.,
I 10
I 1 0 -10
I
I 9
10"
^ i
8
10"
10"
:
1
i 6
10"
—A
>«.
1 10' 5
[Peptide] M
o
0.2
£
-f
,o
7 I
°
" A.
)
I
I
0.2 0.4 0.6 Bound (mol/106 cells 1
1
0.4
0.5
!
0.3
125
I-VIP nM
[VIP] M
FlG. 2. A, Saturation curve and Scatchard analysis of the high affinity VIP binding site in rat granulosa cells. Binding of increasing concentrations of [125I]VIP was measured at 37 C for 30 min. B, Bindinginhibition curve and Scatchard analysis of high and low affinity VIP binding sites. Cells were incubated with [125I]VIP in the presence of increasing concentrations of native VIP. Binding is expressed as a percentage of the [125I]VIP bound in the absence of unlabeled peptide.
FIG. 3. A, Inhibition of [125I]VIP binding by GRF and VIP-related peptides. Granulosa cells were incubated for 30 min at 37 C in the presence of 0.1 nM [125I]VIP and increasing concentrations of VIPrelated and other peptides. Binding is expressed as a percentage of the [125I]VIP bound in the absence of unlabeled peptide. B, Inhibition of [125I][His\Nle27]GRF(l-32)NH2 binding by GRF and VIP-related peptides. Cells were incubated for 45 min at 4 C in the presence of [125I] [His\Nle27]GRF(l-32)NH2 and increasing concentrations of rGRF(l43)OH, [His1,Nle27]GRF(l-32)NH2) VIP, secretin, PHI, GIP, glucagon, /3-endorphin, and CRF. Binding is expressed as a percentage of the [^IHHisSNle27] GRF(1-32)NH2 bound in the absence of unlabeled peptide. Each point represents the mean of three determinations with SE of less than 6%.
M for rGRF, 6.7 x 10~8 M for [His\Nle27]hGRF(l32)NH2, 8.5 X 10"8 M for PHI, and 5.2 x 10"7 M for secretin. GIP, glucagon, and the unrelated peptides, /?endorphin and CRF, did not inhibit [125I]VIP binding. The half-maximum inhibitory concentrations of the VIP-related peptides required to inhibit binding of the [125I]GRF analog differed from those observed with [125I] VIP as tracer, and the relative potencies were: rGRF(l43)OH > [His\Nle27]hGRF(l-32)NH2 > VIP > secretin > PHI (Fig. 3B). The IC50 of rGRF (1 x 10"9 M) was almost 2 orders of magnitude higher than that of VIP,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2121
GRF ACTIONS ON GRANULOSA CELLS
indicating the presence of a GRF-preferring receptor. The unrelated peptides, /3-endorphin and CRF, did not inhibit binding of the radioiodinated GRF analog. The effects of rGRF on cAMP and steroid production, and expression of LH receptors, were studied in basal and FSH-stimulated granulosa cells. GRF (10~7 M) alone stimulated cAMP production as shown by a modest increase in intracellular cAMP levels (Fig. 4A) and a larger rise in extracellular cAMP (Fig. 4B). However, in cells treated with FSH (100 ng/ml), GRF caused substantial elevations of intra- and extracellular cAMP at concentrations of 10~10 to 10"7 M, with ED50 of 10~9 M. The stimulation of cAMP production by GRF was accompanied by parallel enhancement of FSH-induced progesterone (Fig. 5A) and estradiol (Fig. 5B) production. The ability of GRF to amplify the FSH-induced acquisition of steroid biosynthetic capacity and aromatase activity was dose-dependent, with an ED50 of about 10~8 M. Basal progesterone and estradiol levels were unaffected by rGRF treatment.
Basal
FSH 50 ng
FSH 250 ng
FSH 1000 ng
B T 40 30-
T
o
*
^ 20 V> m
10 0
0
rGRF FSH [10-7]M 100 ng
1 0 -o
10
[rGRF] M
-7
1Q -6
Basal
FSH 50 ng
i
_ FSH 250 ng
J
FSH 1000 ng
FIG. 5. Enhancement of FSH-stimulated progesterone (A) and estradiol (B) production by GRF. Granulosa cells (4 x 106 cells/ml) were cultured for 72 h with increasing amounts of FSH in the absence (solid bars) or presence (open bars) of GRF (10~7 M). Each point represents the mean ± SE of triplicate determinations. *,P< 0.05; **, P < 0.01.
The stimulatory effects of GRF on FSH-induced granulosa cell responses were compared with those exerted by VIP. The dose-dependent increases in cAMP accumulation in response to both peptides are shown in Fig. 6A. In terms of the amplification of FSH-induced cAMP accumulation, VIP was more potent and more effective than GRF. The higher potency and efficacy of VIP were also evident in the enhancement of FSH-induced progesterone production (Fig. 6B) and aromatase activity (data LU not shown). The kinetics of the actions of 10~8 M VIP and rGRF on progesterone (Fig. 7A) and estradiol (Fig. v11 1 0 -10 1 0 -9 1Q -8 1Q -7 1Q -6 rGRF FSH 10" 7B) production were determined in cells treated for 72 h [10"7]M 100 ng [rGRF] M with 100 ng oFSH and cultured for a further 48 h in the presence of FSH and both peptides. Compared to conFIG. 4. Stimulation of intracellular (A) and extracellular (B) cAMP accumulation by GRF. Granulosa cells were cultured for 72 h in the trols (FSH alone), both VIP and rGRF increased steroid absence (solid bars) or presence of FSH (100 ng/ml) (hatched bars) and accumulation, and VIP was more effective than rGRF. n 6 increasing concentrations of rGRF (10" to 10" M); C, Control. Each The stimulatory effects of VIP and GRF on expression point represents the mean ± SE of triplicate determinations.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
• VIP
50
f
ArGRF
c ,o o
40 30
3
Endo • 1990 Vol 127 • No 5
80 - A
Pro
(ng/ ml)
2122
/
OFSH
/
/
/
/
/
/
I r
gest eron
-
f
o
//
Oin
J= ^ _L X
8«
300
-
/
XX /^-" /
200
"5 a. Q.
100 H
0
i
250 ng FSH
10" 11
i 10" 10
I 109
/
> r rGRP *
I 10"
I 8
10'
7
fPeptide] M
o
14
_£.
]B i
i
i
• 5 ^ 12 m o 10
al
2
CO
rt
a.
•
•
VIP
GRF
i
o E 4
•1 Control
GRF
VIP
1 FSH
GRF
VIP
GRF
•10 ng-
FIG. 8. Potentiation of FSH-induced LH receptor expression by GRF and VIP. A, Granulosa cells were cultured for 72 h in the presence of
FSH (250 ng/ml) and the indicated concentrations of GRF and VIP. At the end of the incubation, cells were assayed for [125I]hCG binding as described in Materials and Methods. Each point represents the mean ± SE of triplicate determinations. B, Effects of GRF and VIP on LH receptor expression. Granulosa cells were cultured for 72 h with 10~7 M GRF and/or VIP in the absence and presence of 10 ng FSH. Bars indicate the mean ± SE of triplicate determinations. *, P < 0.05.
brain (26), and liver (47, 48). However, only one class of binding sites is present in guinea pig intestinal cells (49) and human peripheral lymphocytes (50). The affinities of the two classes of VIP receptors observed in rat granulosa cells were similar to those reported in other VIP target tissues. There is reasonable agreement between the affinity of the VIP binding sites and the ED50 of VIP-induced responses in granulosa cells in the present and earlier studies (35, 36), as well as in the pituitary [where it stimulates PRL release (51)], in rat gut epithelium, and in the GH3 line of rat pituitary tumor cells (52). A major finding of this study was the potent agonist activity of rGRF(l-43)OH in terms of binding to the VIP receptor and stimulation of cAMP production, similar to its potency in guinea pig pancreas (53). Specific high affinity binding sites for GRF have been described only
2123
in anterior pituitary cells (41, 54, 55) and pituitary membrane homogenates (56), where VIP and GRF receptors are distinct entities with different cellular locations (lactotropes vs. somatotropes) and mechanisms of action, and stringent selectivities for the two peptides. In terms of cAMP production, VIP is a weak agonist at the pituitary level (57) but is a more potent stimulus in the granulosa cell. Studies on the GRF receptor have been facilitated by the use of the potent analog, [125I] [His1,Nle27]hGRF(l-32)NH2, as tracer in the binding assay (41). In rat granulosa cells, the optimal conditions for binding of the 125I-labeled GRF agonist differed markedly from those of [125I]VIP, with maximal specific binding at 4 C rather than 37 C. The level of specific binding of the GRF radioligand was less than that of [125I]VIP, and the relative binding-inhibition potencies for VIP and the GRF peptides were reversed, with rGRF(l-43)OH > [His1,Nle27]GRF(l-32)NH2 > VIP > PHI > secretin. The finding that GRF is almost 1 order of magnitude more potent than VIP in competing with the GRF radioligand for receptor binding suggests that the common receptor for GRF and VIP adopts a GRF-preferring confirmation at low temperature. However, GRF is a relatively less potent agonist than VIP in eliciting biological responses of granulosa cells incubated at 37 C, a difference reflected in its lower activity in [125I] VIP binding-inhibition studies at this temperature. It is likely that the peptides of the VIP/GRF family have evolved through successive events of gene duplication from a single ancestral gene coding for an original peptide (47), with mutations to acquire the structural properties necessary to interact with different sets of receptors. This evolutionary process could be accompanied by changes not only in the peptides, but also in the membrane proteins forming the receptor site. At the ovarian level, as in other target tissues, it is possible that a family of receptors for peptides structurally related to VIP has evolved in parallel with the peptides themselves. The potential functional role of such receptors in the ovary is indicated by the known actions of VIP on granulosa cell development (35-39) and the presence of VIP in the gonads, as noted earlier. It is likely that GRF is also formed in the gonads and acts locally on the VIP receptor. GRF-like material has been detected by RIA in extracts of rat ovary (Moretti C. and A. Bagnato, unpublished data) and in human follicular fluid (16), and by immunostaining in the interstitium of the human ovary (15). Also, a GRF-like factor and its mRNA have been identified in the rat testis (14). GRF itself had only minor effects on granulosa cell function, but potentiated FSH-induced granulosa cell differentiation in a dosedependent manner. The concentrations of GRF required to amplify the stimulatory effects of FSH on granulosa cell activity were consistent with its relative binding
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2124
GRF ACTIONS ON GRANULOSA CELLS
affinity at the VIP/GRF receptor in these cells. It is possible that the presence of adequate concentrations of locally synthethized GRF in the interstitial fluid could modulate granulosa cell responsiveness to gonadotropic stimulation. In infertile patients resistant to gonadotropin therapy, cotreatment with the bioactive N-terminal portion of the peptide (GRF 1-29) enhances the stimulatory action of gonadotropins on ovarian function (16). This effect of exogenous GRF on the human ovary could be exerted through at least two distinct mechanisms. First, the increased production of GH and somatomedin-C (SmC)/ insulin-like growth factor I (IGF-I) would be expected to increase the ovarian response to gonadotropin stimulation. Rat granulosa cells are responsive to GH (58, 59) and SmC/IGF-l (60), both of which potentiate the actions of FSH on differentiation and steroidogenesis. GH does not appear to stimulate the expression of mRNA for SmC/IGF-I or IGF-II in human ovarian tissue (61), but specific receptors for SmC/IGF-I have been recently identified in human granulosa cells (62). Second, the sensitizing action of GRF 1-29 could also be due to a direct action of the peptide on granulosa-cell receptors for peptides of the VIP family, leading to amplification of the actions of FSH on follicle growth and steroidogenic capacity. In the present studies, rGRF caused marked increases in FSH-stimulated cAMP and progestin production, aromatase activity, and expression of LH receptors. These effects were not additive to those of VIP, which caused higher maximal increases in most responses and was about 1 order of magnitude more potent than rGRF. The finding that GRF, as well as VIP, enhances all aspects of granulosa cell differentiation suggests that these peptides can modulate the trophic action of FSH in the control of follicular maturation. The ability of GRF and VIP to enhance FSH-stimulated cAMP production is probably the primary mechanism through which the peptides potentiate granulosa cell differentiation. Although the efficacy of GRF per se is relatively low, its ability to stimulate cAMP production is consistent with the actions of GRF in pituitary somatotrophs (1, 2) and in those peripheral tissues in which GRF recognizes the VIP-receptor, namely human and rat intestinal epithelial cells (63), and rat pancreatic liver membranes (47). The present findings add rGRF to the several peptides known to stimulate the adenylate cyclase-cAMP system in ovarian granulosa cells (64-66). The nature of the dose-response curves for cAMP production and biological actions in granulosa cells indicates that small degrees of VIP/GRF receptor occupancy significantly amplify the FSH-induced adenylate cyclase response. Thus, if FSH is present in low concentrations, the synergistic action of GRF could significantly enhance granulosa-cell responsiveness and accelerate the matu-
Endo • 1990 Vol 127 • No 5
ration process. In conclusion, the receptor-mediated enhancement by GRF of FSH-induced steroidogenesis and LH/hCG receptor expression is probably exerted through the interaction of the peptide with VIP/GRF receptors present on the surface of granulosa cells. The different incubation conditions required for optimal binding of VIP and the GRF-agonist, and the reciprocal affinities of the two structurally related peptides in homologous radioligand binding studies, suggest that the selectivity of the receptor for VIP and GRF is temperature dependent. These findings, and the localization of GRF in ovarian interstitial cells (15), indicate that GRF can exert paracrine regulation, similar to the action of VIP, on ovarian function. In this manner, locally produced or exogenously administered peptide would exert direct positive modulation of trophic hormone-stimulated maturation of the granulosa cell. Since it lacks the hypotensive action of VIP, GRF could provide a therapeutic measure for the activation of ovarian VIP/GRF receptors when local potentiation of gonadotropin action is required for optimal stimulation of ovarian follicular development. Acknowledgments The authors are grateful to Dr. S. Ulisse for helpful discussions and technical advice. This work was supported in part by a grant from Industria Pharmaceutica Serono, Rome, Italy.
References 1. Frohman LA, Jansson GO 1986 Growth hormone releasing hormone. Endocr Rev 7:223 2. Spiess J, Rivier J, Vale W 1983 Characterization of rat hypothalamic growth hormone-releasing factor. Nature 303:532 3. Shibasaki T, Kiyosawa Y, Masuda A, Nakahara M, Imaki T, Wakabayashi I, Demura H, Shizume K, Ling N 1985 Distribution of growth hormone-releasing-factor-like immunoreactivity in human tissues. J Clin Endocrinol Metab 59:263 4. Thorner MO, Vance ML, Evans WS, Ho K, Rogol AD, Blizzard RM, Furlanetto R, Rivier J, Vale W 1986 Growth hormone releasing factor and somatomedin C production: extrahypothalamic localization and possible functional significance. Acta Endocrinol [Suppl] (Copenh) 276:34 5. Ling N, Zeytin F, Bohlen P, Esch F, Brazeau P, Wehrenberg WB, Baird A, Guillemin R 1985 Growth hormone releasing factors. Annu Rev Biochem 54:403 6. Christofides ND, Stephanou A, Suzuki H, Yiangou Y, Bloom SR 1984 Distribution of immunoreactive growth hormone releasing hormone in the human brain and intestine and its production by tumors. J Clin Endocrinol Metab 59:747 7. Bruhn TO, Mason RT, Vale W 1985 Presence of growth-hormonereleasing-factor-like immunoreactivity in rat duodenum. Endocrinology 117:1710 8. Losa M, Wolfram G, Mojto J, Schopohl J, Spiess Y, Huber R, Muller OA, von Werder K 1990 Presence of growth hormonereleasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties. J Clin Endocrinol Metab 70:62 9. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276 10. Nicholson WE, DeCerney GS, Jackson RV, Orth DN 1987 Pitui-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
GRF ACTIONS ON GRANULOSA CELLS
11. 12. 13.
14. 15.
16.
17.
18. 19. 20.
21.
22. 23.
24.
25.
26.
27. 28.
tary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin. Regul Peptides 18:173 Baird A, Wehrenberg WB, Bohlen P, Ling N 1985 Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117:1598 Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098 Margioris NM, Brockmann G, Bohler Jr HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 126:151 Berry SA, Pescovitz OH 1988 Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 123:661 Moretti C, Fabbri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazzicone Q, Spera G 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads. J Endocrinol Invest 13:301 Moretti C, Fabbri A, Gnessi L, Forni L, Fraioli F, Frajese G 1989 GHRH stimulates follicular growth and amplified FSH-induced ovarian folliculogenesis in women with anovulatory infertility. In: Frajese G, Steinberg E, Rodriguez-Rigau LJ (eds) Reproductive Medicine: Medical Therapy. Proceedings of the Second International Symposium on Reproductive Medicine. Elsevier Science Publishers, New York, no 875, pp 103-112 Christophe J, Svoboda M, Dehaye JP, Winand J, VandermeersPire MC, Vandermeers A, Cauvin A, Gourlet P, Robberecht P 1989 The VIP/PHI/secretin/helodermin/helospectin/GRF family: structure-function relationship of the natural peptides, their precursors and synthetic analogues as tested in vitro on receptors and adenylate cyclase in a panel of tissue membranes. In: Martinez J (ed) Peptide Hormones as Prohormones: Processing, Biological Activity, Pharmacology. Halsted Press, New York, pp 211-242 Bataille D 1986 VIP and related substances. Peptides 7 [Suppl 1] 1:390 Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides 7 [Suppl 1] 1:27 Schwartz CJ, Kimberg DV, Sheerin HE, Field M, Said SI 1974 Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54:536 Feliu JE, Mojena M, Silvestre RA, Monge L, Marco J 1983 Stimulatory effect of VIP on glycogenolysis and gluconeogenesis in isolated rat hepatocytes: antagonism by insulin. Endocrinology 112:2120 Said SI, Mutt V 1970 Polypeptide with broad biological activity: isolation from small intestine. Science 169:1217 Bataille D, Freychet P, Rosselin G 1974 Interactions of glucagon, gut glucagon, vasoactive intestinal polypeptide and secretin with liver and fat cell plasma membranes: binding to specific sites and stimulation of adenylate cyclase. Endocrinology 95:713 Bataille D, Besson J, Bastard C, Laburthe M, Rosselin G 1977 Specificity in hormone-receptor interaction: studies with insulin, glucagon and vasoactive intestinal peptide (VIP). In: Bonfils S, Fromageot P, Rosselin G (eds) Hormonal Receptors in Digestive Tract Physiology. North Holland, Amsterdam, p 113-119 Dharmsathaphorn K, Harms V, Binder HJ, Yamashiro DJ, Wright EM 1983 Preferential binding of vasoactive intestinal polypeptide to basolateral membrane of rat and rabbit enterocytes. J Clin Invest 75:462 Staun-Olsen P, Ottesen B, Bartels D, Nielsen MH, Gammeltoft S, Fahrenkrug J 1982 Receptors for vasoactive intestinal polypeptide on isolated synaptosomes from rat cerebral cortex. Heterogeneity of binding and desensitization of receptors. J Neurochem 39:1242 Christophe J, Conlon TP, Gardner JD 1976 Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. J Biol Chem 251:4629 Robberecht P, Waelbroeck M, Camus JC, DeNeef P, Christophe J 1984 Importance of disulfide bonds in receptors for vasoactive
29. 30.
31. 32. 33. 34.
35. 36.
37.
38.
39.
40. 41. 42. 43. 44.
45. 46. 47. 48.
49.
2125
intestinal peptide and secretin in rat pancreatic plasma membranes. Biochim Biophys Acta 773:271 Prieto JC, Carmena MJ 1983 Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate. Biochim Biophys Acta 763:408 Zhen-Guo L, Queen G, LaBella FS 1990 Adrenocorticotropin, vasoactive intestinal polypeptide, growth hormone-releasing factor, and dynorphin compete for common receptors in brain and adrenal. Endocrinology 126:1327 Wanke IE, Rorstad OP 1990 Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: localization of binding to lactotropes. Endocrinology 126:1981 Larson LI, Fahrenkrug J, Schaffalitzky de Muckadell OB 1977 Vasoactive intestinal polypeptide occurs in nerves of the female genitourinary tract. Science 197:1374 Aim P, Alumets J, Hakanson R, Helm G, Owman C, Sjoberg NO, Sundeler F 1980 Vasoactive intestinal polypeptide nerves in the human female genital tract. Am J Obstet Gynecol 136:349 Klein CM, Burden HW 1988 Substance P and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers in relation to ovarian postganglionic perikarya in para- and prevertebral ganglia: evidence from combined retrograde tracing and immunocytochemistry. Cell Tissue Res 252:403 Davoren BJ, Hsueh AJW 1985 Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. Biol Reprod 33:37 Trzeciak WH, Ahmed CE, Simpson ER, Ojeda SR 1986 Vasoactive intestinal peptide induces the synthesis of the cholesterol sidechain cleavage enzyme complex in cultured rat ovarian granulosa cells. Proc Natl Acad Sci USA 83:7490 George FW, Ojeda SR 1987 Vasoactive intestinal peptide enhances aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone. Proc Natl Acad Sci USA 84:5803 Tornell J, Carlsson B, Hillensjo T 1988 Vasoactive intestinal peptide stimulates oocyte maturation, steroidogenesis and cyclic adenosine 3',5' monophosphate production in isolated preovulatory rat follicles. Biol Reprod 39:213 Liu YX, Kasson BG, Dahl KD, Hsueh AJW 1987 Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes. Peptides 8:29 Gozes I, Tsafriri A 1986 Detection of vasoactive intestinal peptideencoding messenger ribonucleic acid in the rat ovaries. Endocrinology 119:2606 Seifert H, Perrin M, Rivier J, Vale W 1985 Binding sites for growth hormone releasing factor on rat anterior pituitary cells. Nature 313:487 Knecht M, Katz MS, Catt KJ 1981 Gonatropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells. J Biol Chem 256:34 Catt KJ, Dufau ML 1975 Gonadal receptors for LH and chorionic gonadotropin. Methods Enzymol 37:167 Christophe J, Svoboda M, Lambert M, Waelbroeck M, Winand J, Dehaye JP, Vandermeers-Piret MC, Vandermeers A, Robberecht P 1986 Effector mechanism of peptides of the VIP Family. Peptides 7 [Suppl l]:101 Laburthe M, Couvineau A 1988 Molecular analysis of vasoactive intestinal peptide receptors. A comparison with receptors for VIPrelated peptides. Ann NY Acad Sci 527:296 Laburthe M, Breant B, Rouyer-Fessard C 1984 Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Eur J Biochem 139:181 Christophe J, Svoboda M, Magali W, Winand J, Robberecht P 1988 Vasoactive intestinal peptide receptors in pancreas and liver. Ann NY Acad Sci 527:238 Couvineau AB, Amiranoff B, Laburthe M 1986 Solubilization of the liver VIP receptor: hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein. J Biol Chem 261:8527 Binder HJ, Lemp GF, Gardner JP 1980 Receptors for vasoactive intestinal peptide and secretin in small intestinal epithelial cells.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
2126
GRF ACTIONS ON GRANULOSA CELLS
Am J Physiol 238:190 50. Danek A, O'Dorisio MS, O'Dorisio TM, George JM 1983 Specific binding sites for vasoactive intestinal polypeptide on nonadherent peripheral blood lymphocytes. J Immunol 131:1173 51. Rotsztejn WH, Dussaillant M, Nobou F, Rosselin G 1981 Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture. Proc Natl Acad Sci USA 78:7584 52. Zeytin FN, Reyl-Desmars F, Rathbun T 1985 Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP-prefering receptor site(s). Biochem Biophys Res Comraun 127:992 53. Pandol SJ, Seifert H, Thomas MW, Rivier J, Vale W 1984 Growth hormone-releasing factor stimulates pancreatic enzyme secretion. Science 225:326 54. Valicelebi G, Santacroce TM, Harpold MM 1985 Specific binding of synthetic human pancreatic growth hormone releasing factor (140 OH) to bovine anterior pituitaries. Biochem Biophys Res Commun 126:33 55. Ikuyama S, Natori S, Nawata H, Kato KI, Ibayashi H, Kariya T, Sakai T, Rivier J, Vale W 1988 Characterization of growth hormone-releasing hormone receptors in pituitary adenomas from patients with acromegaly. J Clin Endocrinol Metab 66:1265 56. Seifert H, Perrin M, Rivier J, Vale W 1985 Growth hormonereleasing factor binding sites in rat anterior pituitary membrane homogenate: modulation by glucocorticoids. Endocrinology 117:424 57. Magistretti PJ, Schoenberg P, Kehrer P, Martin JL, Gaillard RC 1986 Actions of VIP, hGRF, PHI and Secretin: comparative studies in cerebral cortex and adenohypophysis. Peptides 7[Suppl 1]:175 58. Jia XC, Kalmijn J, Hsueh AJW 1986 Growth hormone enhances
59. 60.
61.
62. 63.
64.
65. 66.
Endo • 1990 Vol 127 • No 5
follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 118:1601 Davoren JB, Hsueh AJW 1986 Growth hormone increases ovarian levels of immunoreactive somatomedin C/insulin-like growth factor I in vivo. Endocrinology 118:888 Adashi EY, Resnick CE, Brodie AMH, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells. Endocrinology 117:2313 Voultilainen R, Miller WL 1987 Coordinate tropic hormone regulation of mRNAs for insulin-like growth factor II and the cholesterol side chain cleavage enzyme, P450scc, in human steroidogenic tissue. Proc Natl Acad Sci USA 84:1590 Poretsky L, Kalin MF 1987 The gonadotropic function of insulin. Endocr Rev 8:132 Laburthe M, Couvineau A, Rouyer-Fessard C 1986 Study of species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal peptide (VIP) receptors using GRF and intestinal VIP receptors from rat and human: evidence that AcTyr1 hGRF is a competitive VIP antagonist in the rat. Mol Pharmacol 29:23 Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1986 Somatomedin-C as an amplifier of follicle-stimulating hormone action: enhanced accumulation of adenosine 3',5'-mono phosphate. Endocrinology 118:149 Knecht M, Catt KJ 1983 Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 258:2789 Knecht M, Feng P, Catt KJ 1989 Transforming growth factorbeta: autocrine, paracrine, and endocrine effects in ovarian cells. Semin Reprod Endocrinol 1:12
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:26 For personal use only. No other uses without permission. . All rights reserved.
