The Porcine Ovarian Follicle: III. Development of Chorionic Gonadotropin Receptors Associated with Increase in Adenyl Cyclase Activity During Follicle Maturation C. Y. LEEf Department of Molecular Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 ABSTRACT. Specific binding of human chorionic gonadotropin (hCG), and the hCG-sensitive adenyl cyclase of granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) porcine ovarian follicles have been studied. The number of hCG-binding sites per cell (n) increases during follicle maturation without a change in the binding affinity. The values for n were 300-400 for small, 1,000-1,600 for medium, and 8,20010,000 for large-follicle cells. The dissociation constant is 2.4 x 10~IOM for all cells. hCG-sensitive adenyl cyclase was demonstrated in porcine granulosa cells. The adenyl cyclase system of granulosa cells becomes increasingly responsive to hCG stimulation during follicle development. Maximal
R
adenyl cyclase activation by hCG (1 /xg/ml) was 240, 750, and 7,000 molecules of cyclic AMP formed/sec/cell, respectively, for small, medium, and large follicle cells. The concentration of hCG giving half-maximal stimulation (1.0 X 10~DM) was similar for both large and medium follicle cells. It is concluded that: 1) an increase in hCG receptor sites per cell occurs during maturation of the porcine ovarian follicle without change of binding affinity, and 2) the increase in the number of hCG receptors correlates well with hCG-sensitive adenyl cyclase activity during follicle development. (Endocrinology 99: 42, 1976)
The present investigation was undertaken to characterize the hCG-receptor interaction of granulosa cells and to correlate the development of hCG receptors with the hCGsensitive adenyl cyclase activity of granulosa cells during follicle maturation.
ECEPTORS specific for hCG-LH have been demonstrated in the rat ovaiy (1,2) and testis (3). Gonadotropin receptors have also been reported in porcine granulosa cells (4). This intact cell system has proved useful for the direct study of gonadotropin-receptor interaction, since granulosa cells are a homogeneous cell population and easily obtained without enzymatic treatment. Channing and Kammerman (5) have reported that granulosa cells obtained from large follicles bound more [125I]iodohCG than medium and small follicles. Very recently, it has been shown that increased gonadotropin-binding activity is due to an increased number of receptor sites per cell and not to increased binding affinity (6,7).
Materials and Methods Granulosa cells Ovaries were obtained from 4 to 6-monthold pigs at a local slaughterhouse within 20 minutes of death. The ovaries were placed immediately in ice-cold 0.9% NaCl and brought back to the laboratory within 2 - 3 hours. Granulosa cells were harvested from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) ovarian follicles; according to the method of channing (8). The cells were washed twice, centrifuged, and resuspended in cold medium 199 with Earle's salts, pH 7.4 (Grand Island Biological Co.). The number of cells were counted in 0.04% erythrosin B in phosphate-buffered saline using a hemocytometer.
Received November 12, 1975. Supported by Grants from USPHS (HD 08099 and HD 9140) and the Mayo Foundation. Part of this work was presented at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Canada, June 2-5, 1975. t Present Address: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.
Assay ofhCG binding The procedure for iodination of hCG to approximately 1 atom 125I per molecule of hCG 42
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hCG RECEPTORS OF GRANULOSA CELLS with full binding activity was described previously (9). Highly purified hCG (CR117) was a gift of Dr. Robert Canfield, Columbia University. Highly purified hLH (22870-1B) was obtained from Dr. R. J. Ryan, Mayo Clinic (10). Ovine FSH-S7 and ovine prolactin-S6 were obtained from the National Institutes of Health, Bethesda, MD. The typical assay mixture consisted of 2-10 x 106cells, 5 ng [l25I]iodohCG or other test substances, in a final volume of 0.5 ml of 85% Medium 199 with Earle's salts (pH 7.4) containing 15% immature porcine serum. Porcine serum was added to prevent the aggregation of cells when incubation was carried out over 2 h. The incubation was at 37 C with constant shaking (90 cycles/m in). At the end of incubation, 4.0 ml of ice-cold Tris buffered saline (pH 7.4) containing 1% bovine serum albumin was added to the assay tubes to stop the reaction. The tubes were immediately centrifuged at 2,000 rpm for 5 min in an International Model PR-6 centrifuge. The cell pellet was suspended and washed twice. The radioactivity bound to the cells was measured in a gamma spectrometer. Binding in the presence of a large excess of unlabeled hCG (200 IU per tube) was used to determine nonspecific binding. Assessment ofhCG inactivation Labeled hCG (2 ng/tube) was incubated with medium follicle cells (10 x 106/tube) in Medium 199 containing 0.1% bovine serum albumin. The incubation was at 37 C for various periods of time. The incubation medium was separated from cells by centrifugation at the end of incubation, and the labeled hCG contained in the supernatant was assessed for its ability to bind to fresh granulosa cells. As control, labeled hCG was incubated under the same conditions, but without cells. Adenyl cyclase assay The cells kept in Medium 199 with Earle's salts were washed with 25 mM Tris buffered 0.9% NaCl and resuspended in ice-cold medium containing 27% (wt/voJ) sucrose, 1.0 mM EDTA and 10 mM Tris-HCl, pH 7.5 (11). The homogenate was prepared in a Teflon-glass homogenizer using 3 strokes in a final concentration of 160 x 106 cells/ml. Twenty-five /xl aliquots (4 x 106 cells) of homogenate were used for adenyl cyclase assay.
43
Adenyl cyclase activity was determined according to the method described by HunzickerDunn and Birnbaumer (12) with minor modification. The incubation mixture contained 25 mM Tris-HCl, pH 7.0, 5.0 mM MgCl2) 1 mM EDTA, 1 mM 3-isobutyl-l-methylxanthine, an ATP-regenerating system consisting of 20 mM creatine phosphate and 0.2 mg/ml of creatine kinase at pH 7.0, 1 mM cyclic AMP, 3 mM [a-32P]ATP (40-50 cpm/pmole), and hCG as indicated. The reaction was initiated by the addition of cell homogenates to give 4 x 106 cells per tube. The final volume of the reaction was 50 fx\. Incubation was at 30 C for 20 min. The reaction was terminated by the addition of 0.1 ml of "stopping solution" containing 40 mM ATP, 12.5 mM cyclic AMP, [3H]cyclic AMP (approximately 15,000 cpm), followed by heating at 100 C for 3 min. Cyclic AMP was isolated acording to the method of Salomon et al. (13) using Dowex and alumina chromatography. The final eluate (4.5 ml) was collected directly into scintillation vials containing 15 ml Aquasol. The radioactivity present in the eluate containing [32P]cyclic AMP and [3H]cyclic AMP was determined in a Beckman LS-100C liquid scintillation counter. Cyclase activity is expressed as picomoles cyclic AMP formed per 20 min per nig protein. Protein was determined by the procedure of Lowry et al. (14) after the digestion of homogenates with O.lN NaOH at 50 C for 30 min. Crystalline bovine serum albumin was used as a standard.
Results Properties of [125I]iodo-hCG binding granulosa cells
to
Figure 1 shows that the binding of [125I]iodo-hCG to granulosa cells is dependent on incubation time, temperature, and the number of cells. At 37 C, equilibrium was reached within 5-6 h. The binding reaction at 25 C proceeded at a slower rate. Total binding amounted to 31 and 22% of the total radioactivity with 5 x 106 and 2 x 106 cells/tube, respectively, using cells from large follicles. Nonspecific binding (that measured in the presence of an excess of unlabeled hCG) constituted only 0.20.5% of the total radioactivity and 1-2% of the total binding. The amount of labeled
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Endo • 1976 Vol 99 • No 1
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44
37°C
FIG. 1. Effect of incubation time and temperature on [l25I]iodohCG binding to large follicle cells. Binding assay was performed as described in Methods. [125I]iodo-hCG was 5 ng/tube. (•) 5 x 106 cells/tube; (A,X) 2 x 106 cells/tube.
0
2
4
6
Incubation time, hours
hCG bound showed a linear relationship to the number of cells used up to 10 x 106 cells/tube (Fig. 2). The specificity of hCG binding to granulosa cells is shown in Fig. 3. Both unlabeled hCG and hLH were equally potent in inhibiting labeled hCG binding, with 50% displacement at 8 ng per tube. Ovine FSH and prolactin did not significantly inhibit binding at doses as high as 10 jug/tube.
Only slight inactivation of [125I]iodo-hCG occurred during the incubation with granulosa cells, based on the capacity of labeled hCG for rebinding to fresh granulosa cells. After incubation for 0.5, 1, and 2h at 37 C, [125I]iodo-hCG retained 96%, 93%, and 90% binding activity compared with the control which was incubated in the buffer without cells, as described in Methods. Quantitative binding
28
1 1
24 20 16 12 8
2 4 6 8 Number of cells (10~6/tube)
10
FIG. 2. Effect of number of cells on [125I]iodohCG binding to medium follicle cells. Incubation was at 37 C for 1 h.
study
of [}25I]iodo-hCG
Granulosa cells from 3 sizes of follicles were incubated with increasing amounts of [125I]iodo-hCG at 37 C for 5-6 h, at which time binding activity had reached a plateau. The binding of hCG to medium follicle cells as a function of hCG concentrations is shown in Fig. 4. Nonspecific binding was linearly related to the concentration of [125I]iodo-hCG. A saturable specific binding curve was obtained when nonspecific binding was subtracted from total binding (Fig. 4). The Scatchard plot of the specific binding data for the medium follicle cells is illustrated in Fig. 5 (middle curve). The equilibrium dissociation con-
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45
hCG RECEPTORS OF GRANULOSA CELLS
100
80 \ FlG. 3. Displacement of [l25I]iodo-hCG binding to medium follicle cells by unlabeled hormones. Incubation was at 37 C for l h ; [125I]iodo-hCG was 5 ng/tube.
. 60
40
20
1
10'
10z
Unlabeled hormone,
stant, calculated from the Scatchard plot, is 2.1 x 10~10M. The number of binding sites for medium follicle cells was estimated to be 16.9 x 10~15 moles per 10 x 106 cells, which is equivalent to approximately 1,000 hCG molecules per cell. The Scatchard plots of the specific binding data for small follicle cells (left curve) and large follicle cells (right curve) are also illustrated in Fig. 5. It must be noted that 10 x 106 cells/tube were used for both small and medium follicle cells, while only
10 3
10"
ng/tube
2 x 106 cells/tube were used for large follicle cells. The data for dissociation constants and the number of receptor sites per cell for granulosa cells from follicles at various stages of development are compared in Table 1. The values of the dissociation constants for small, medium, and large follicle cells are similar, and average 2.4 x 10~10M. The number of binding sites per cell, however, increases 4-fold from
small to medium follicle cells and 26-fold from small to large follicle cells. Total binding
50
FIG. 4. Effect of [125I]iodohCG concentration on [l25I]iodohCG binding to medium follicle cells. Incubation was at 37 C for 5 h with 10 x 106 cells/ tube.
20
40
[
IZ5
60
80
l] hCG, ng/ml
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Endo • 1976 Vol 99 • No 1
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46
TABLE 1. Dissociation constants (IQi) and numbers of binding sites (n) for the granulosa cells of small, medium, and large follicles Granulosa cells of follicles
M x 10'°
n (molecules hCG/cell)
Small (1-2 mm)
1.6 2.5
300 400
Medium (3-5 mm)
2.1 3.8 2.4
1,000 1,400 1,600
Large (6-12 mm)
2.5 1.7
8,200 10,200
hCG-sensitive adenyl cyclase activity Under the assay conditions (3 mM ATP and 5 mM added MgCl2), the adenyl cyclase activities of granulosa cell homogenates in both the absence and the presence of a saturating concentration of hCG were linearly related to the incubation time up to 20 min at 30 C and were also proportional to the number of cells used, up to 4 x 106 cells/tube (unpublished observations). Figure 6 shows that basal adenyl cyclase activity appeared to be higher in large follicle cells. hCG at a concentration 24
as low as 4 ng/ml was sufficient to cause adenyl cyclase activity of large follicle cells to increase 1.8-fold compared with basal activity. The maximal stimulatory response (13-fold relative to basal activity) of large follicle cells was reached at 1 /ag/ml of hCG (Fig. 6). the apparent concentration which gave half-maximal stimulation (apparent Ks) was 45 ng/ml or 9.8 x 10~10M. The inhibition of cyclase activity at 10 /Ag/ml hCG, shown in Fig. 6, was not frequently observed. Both medium and small follicle cells were much less responsive to hCG stimulation with respect to adenyl cyclase activity. hCG at the maximal stimulating dose (1 /xg/ml) caused only 3.7-fold increase in adenyl cyclase activity of medium follicle cells. The apparent Ks (53 ng/ml) is similar to the value obtained for large follicle cells. Small follicle cells were the least responsive to hCG stimulation. Only a very small increment (1.5-fold) of adenyl cyclase activity was obtained at the concentration of 1 /xg/ ml. The limited response of small follicle cells does not allow an accurate estimation of apparent Ks.
-
FIG. 5. Scatchard plots of [ 125 I]iodo-hCG binding to granulosa cells. Incubation was at 37 C for 5-6 h. (X) small follicle cells, 10 x 106 cells/ tube; (•) medium follicle cells, 10 x 108 cells/tube; (A) large follicle cells, 2 x 106 cells/tube.
8
12
Bound (10"
i5
16
20
24
28
mol/tube)
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47
hCG RECEPTORS OF GRANULOSA CELLS Discussion In previous reports it was demonstrated that LH receptors in the rat ovary can be induced by pregnant mare's serum gonadotropin and hCG priming. The induced LH receptors in the pseudopregnant rat ovary did not differ from those in the immature ovaiy in terms of affinity and specificity for hLH binding (9). The present study indicates similar phenomena. The increase of hCG receptors in porcine granulosa cells during follicle maturation was not accompanied by a change in binding affinity (or dissociation constant). The same finding was also recently reported for porcine granulosa cells by Kammerman and Ross (7). Soil et al. (15) have demonstrated that the decreased insulin receptors in the liver membrane of the ob/ob mouse are indistinguishable from normal with respect to binding affinity, kinetic association and dissociation, temperature dependence, and biological specificity of the binding reaction. It thus appears that the modulation of target tissue response to polypeptide hormone during various functional states is by means of change in the number of hormone receptor sites and not in the binding affinity. The values of the dissociation constants for hCG binding to small, medium, and large follicle cells are in good agreement with the data reported for the hCG-ovarian receptor interaction (16). The mean value of the dissociation constant (2.4 x 10~ 10 M) for granulosa cells from three sizes of
400
f 200
100
100
1000 10,000
100,000
hCG (ng/ml)
FIG. 6. Dose-response curves for hCG-stimulated adenyl cyclase activity of granulosa cells from large, medium, and small follicles. Cyclase assay was performed as described in the Methods. Vertical line indicates the hCG concentration giving half-maximal stimulation of cyclase activity.
follicles compares favorably with the mean value of 1.7 x 10~10M reported by Kammerman and Ross (7). Since little inactivation of free labeled hCG occurred during incubation with intact cells, an accurate estimation of the dissociation constant can be reasonably achieved without correction for inactivation. The number of hCG receptor sites per cell obtained in this study is also in good agreement with reported values (7). The present study also demonstrates the presence of hCG-sensitive adenyl cyclase in porcine granulosa cells and the close
TABLE 2. Comparison of maximal hCG-stimulated adenyl cyclase activity* and the number of hCG receptor sites of granulosa cells cAMP formed 10~12 mole/20 min
cAMP formed molecules/sec
Number off receptors
cAMP formed molecules/sec
Granulosa cells of follicles
4 x 106 cell
cell
cell
receptor
small medium large
1.9 6.0 55.9
240 750 7,000
350 1,300 9,200
0.69 0.58 0.76
* Adenyl cyclase activity was measured according to the Methods at the maximal stimulating concentration (lMg/ml)ofhCG. f Mean values taken from Table 1.
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48
Endo • 1976 Vol 99 • No 1
LEE
correlation between the number of hCG receptor sites and hCG-stimulated adenyl cyclase activity during granulosa cell differentiation. The increase in hCG receptor sites from small to medium follicle cells and the further increase in receptor sites to large follicle cells was accompanied by a proportional increase in hCGsensitive adenyl cyclase activity. These results are more clearly illustrated in Table 2, in which adenyl cyclase activity was calculated from the maximal stimulatory response of granulosa cells at 1 /ug/ml of hCG. The number of cyclic AMP molecules formed per second per cell is proportional to the number of receptor sites per cell for all granulosa cells. This is indicated by the relatively constant ratio (0.58-0.76) of the number of cyclic AMP molecules formed per molecule of hCG receptor. This result does not imply that there is a proportional relationship between the receptor occupancy and the activity of the catalytic subunit of adenylate cyclase. Table 2 only illustrates that the maximal adenyl cyclase activated at the particular hCG concentration studied (1 jug/ml) is dependent on the number of hCG receptor sites available. Enhanced activity of catalytic subunits of the adenyl cyclase of granulosa cells during follicle maturation probably does not occur, as evidenced by the observations that there was no difference in adenyl cyclase activation by NaF and by Prostaglandins Ej and E2 among small, medium, and large follicle cells (unpublished data). Channing and Kammerman (5) have reported an increase in the hCG-binding activity of porcine granulosa cells during follicle development. The enhanced binding activity of large follicle cells was shown to correlate with increases in cyclic AMP production and progesterone synthesis (8). Scanning electron microscopy studies revealed unique changes of the plasma membrane surface of granulosa cells during
follicle development (Chang, S. C., W. Anderson, C. Y. Lee, and R. J. Ryan, manuscript in preparation). Massive numbers of microvilli appeared on large follicle cell surfaces, in contrast to the relatively smooth cell surfaces of small follicle cells. Although the size of granulosa cells from all sizes of follicles has been reported to remain constant (5), the increased number of binding sites per cell does not necessarily imply an increase in binding sites per unit of surface area, since cell surface area may increase without change in cell size. Whether the newly developed hCG receptor sites of large follicle cells are on the microvilli needs to be determined. References 1. Lee, C. Y., and R. J. Ryan, Endocrinology 89: 1515, 1971. 2. Danzo, B. J., A. R. Midgley, and L. J. Kleinsmith, Proc Soc Exp Biol Med 129: 88, 1972. 3. Catt, K. J., M. L. Dufau, and T. Tsuruhara, J Clin Endocrinol Metab 37: 860, 1971. 4. Kammerman, S., R. E. Canfield, J. Kolena, and C. P. Channing, Endocrinology 91: 65, 1972. 5. Channing, C. P., and S. Kammerman, Endocrinology 92: 531, 1973. 6. Lee, C. Y., In Vitro 10: 343, 1974 (Abstract). 7. Kammerman, S., and J. Ross, J Clin Endocrinol Metab 41: 546, 1975. 8. Channing, C. P., and F. Ledwitz-Rigby, In Hardman, J. G., and B. W. O'Malley (eds.), Methods of Enzymology, vol. 39, Academic Press, New York, 1975, p. 183. 9. Lee, C. Y., and R. J. Ryan, Biochemistry 12: 4609, 1973. 10. Ryan, R. J. J Clin Endocrinol Metab 28: 886,1968. 11. Birnbaumer, L., and Po-Chang Yang, J Biol Chem 249: 7867, 1974. 12. Hunzicker-Dunn, M., and L. Birnbaumer, Endocrinology (In press). 13. Salomon, Y., C. Londos, and M. Rodbell, Anal Biochem 58: 541, 1974. 14. Lowry, O. H., N. Y. Rosebrough, A. L. Farr, and R. J. Randall J Biol Chem 193: 265, 1951. 15. Soil, A. H., and C. R. Kahn, J Biol Chem 250: 4702, 1975. 16. Lee, C. Y., and R. J. Ryan, In Endocrine Function of the Human Ovary, Serono Symposium, Florence, 1975 (In press).
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R
adenyl cyclase activation by hCG (1 /xg/ml) was 240, 750, and 7,000 molecules of cyclic AMP formed/sec/cell, respectively, for small, medium, and large follicle cells. The concentration of hCG giving half-maximal stimulation (1.0 X 10~DM) was similar for both large and medium follicle cells. It is concluded that: 1) an increase in hCG receptor sites per cell occurs during maturation of the porcine ovarian follicle without change of binding affinity, and 2) the increase in the number of hCG receptors correlates well with hCG-sensitive adenyl cyclase activity during follicle development. (Endocrinology 99: 42, 1976)
The present investigation was undertaken to characterize the hCG-receptor interaction of granulosa cells and to correlate the development of hCG receptors with the hCGsensitive adenyl cyclase activity of granulosa cells during follicle maturation.
ECEPTORS specific for hCG-LH have been demonstrated in the rat ovaiy (1,2) and testis (3). Gonadotropin receptors have also been reported in porcine granulosa cells (4). This intact cell system has proved useful for the direct study of gonadotropin-receptor interaction, since granulosa cells are a homogeneous cell population and easily obtained without enzymatic treatment. Channing and Kammerman (5) have reported that granulosa cells obtained from large follicles bound more [125I]iodohCG than medium and small follicles. Very recently, it has been shown that increased gonadotropin-binding activity is due to an increased number of receptor sites per cell and not to increased binding affinity (6,7).
Materials and Methods Granulosa cells Ovaries were obtained from 4 to 6-monthold pigs at a local slaughterhouse within 20 minutes of death. The ovaries were placed immediately in ice-cold 0.9% NaCl and brought back to the laboratory within 2 - 3 hours. Granulosa cells were harvested from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) ovarian follicles; according to the method of channing (8). The cells were washed twice, centrifuged, and resuspended in cold medium 199 with Earle's salts, pH 7.4 (Grand Island Biological Co.). The number of cells were counted in 0.04% erythrosin B in phosphate-buffered saline using a hemocytometer.
Received November 12, 1975. Supported by Grants from USPHS (HD 08099 and HD 9140) and the Mayo Foundation. Part of this work was presented at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Canada, June 2-5, 1975. t Present Address: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.
Assay ofhCG binding The procedure for iodination of hCG to approximately 1 atom 125I per molecule of hCG 42
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hCG RECEPTORS OF GRANULOSA CELLS with full binding activity was described previously (9). Highly purified hCG (CR117) was a gift of Dr. Robert Canfield, Columbia University. Highly purified hLH (22870-1B) was obtained from Dr. R. J. Ryan, Mayo Clinic (10). Ovine FSH-S7 and ovine prolactin-S6 were obtained from the National Institutes of Health, Bethesda, MD. The typical assay mixture consisted of 2-10 x 106cells, 5 ng [l25I]iodohCG or other test substances, in a final volume of 0.5 ml of 85% Medium 199 with Earle's salts (pH 7.4) containing 15% immature porcine serum. Porcine serum was added to prevent the aggregation of cells when incubation was carried out over 2 h. The incubation was at 37 C with constant shaking (90 cycles/m in). At the end of incubation, 4.0 ml of ice-cold Tris buffered saline (pH 7.4) containing 1% bovine serum albumin was added to the assay tubes to stop the reaction. The tubes were immediately centrifuged at 2,000 rpm for 5 min in an International Model PR-6 centrifuge. The cell pellet was suspended and washed twice. The radioactivity bound to the cells was measured in a gamma spectrometer. Binding in the presence of a large excess of unlabeled hCG (200 IU per tube) was used to determine nonspecific binding. Assessment ofhCG inactivation Labeled hCG (2 ng/tube) was incubated with medium follicle cells (10 x 106/tube) in Medium 199 containing 0.1% bovine serum albumin. The incubation was at 37 C for various periods of time. The incubation medium was separated from cells by centrifugation at the end of incubation, and the labeled hCG contained in the supernatant was assessed for its ability to bind to fresh granulosa cells. As control, labeled hCG was incubated under the same conditions, but without cells. Adenyl cyclase assay The cells kept in Medium 199 with Earle's salts were washed with 25 mM Tris buffered 0.9% NaCl and resuspended in ice-cold medium containing 27% (wt/voJ) sucrose, 1.0 mM EDTA and 10 mM Tris-HCl, pH 7.5 (11). The homogenate was prepared in a Teflon-glass homogenizer using 3 strokes in a final concentration of 160 x 106 cells/ml. Twenty-five /xl aliquots (4 x 106 cells) of homogenate were used for adenyl cyclase assay.
43
Adenyl cyclase activity was determined according to the method described by HunzickerDunn and Birnbaumer (12) with minor modification. The incubation mixture contained 25 mM Tris-HCl, pH 7.0, 5.0 mM MgCl2) 1 mM EDTA, 1 mM 3-isobutyl-l-methylxanthine, an ATP-regenerating system consisting of 20 mM creatine phosphate and 0.2 mg/ml of creatine kinase at pH 7.0, 1 mM cyclic AMP, 3 mM [a-32P]ATP (40-50 cpm/pmole), and hCG as indicated. The reaction was initiated by the addition of cell homogenates to give 4 x 106 cells per tube. The final volume of the reaction was 50 fx\. Incubation was at 30 C for 20 min. The reaction was terminated by the addition of 0.1 ml of "stopping solution" containing 40 mM ATP, 12.5 mM cyclic AMP, [3H]cyclic AMP (approximately 15,000 cpm), followed by heating at 100 C for 3 min. Cyclic AMP was isolated acording to the method of Salomon et al. (13) using Dowex and alumina chromatography. The final eluate (4.5 ml) was collected directly into scintillation vials containing 15 ml Aquasol. The radioactivity present in the eluate containing [32P]cyclic AMP and [3H]cyclic AMP was determined in a Beckman LS-100C liquid scintillation counter. Cyclase activity is expressed as picomoles cyclic AMP formed per 20 min per nig protein. Protein was determined by the procedure of Lowry et al. (14) after the digestion of homogenates with O.lN NaOH at 50 C for 30 min. Crystalline bovine serum albumin was used as a standard.
Results Properties of [125I]iodo-hCG binding granulosa cells
to
Figure 1 shows that the binding of [125I]iodo-hCG to granulosa cells is dependent on incubation time, temperature, and the number of cells. At 37 C, equilibrium was reached within 5-6 h. The binding reaction at 25 C proceeded at a slower rate. Total binding amounted to 31 and 22% of the total radioactivity with 5 x 106 and 2 x 106 cells/tube, respectively, using cells from large follicles. Nonspecific binding (that measured in the presence of an excess of unlabeled hCG) constituted only 0.20.5% of the total radioactivity and 1-2% of the total binding. The amount of labeled
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Endo • 1976 Vol 99 • No 1
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44
37°C
FIG. 1. Effect of incubation time and temperature on [l25I]iodohCG binding to large follicle cells. Binding assay was performed as described in Methods. [125I]iodo-hCG was 5 ng/tube. (•) 5 x 106 cells/tube; (A,X) 2 x 106 cells/tube.
0
2
4
6
Incubation time, hours
hCG bound showed a linear relationship to the number of cells used up to 10 x 106 cells/tube (Fig. 2). The specificity of hCG binding to granulosa cells is shown in Fig. 3. Both unlabeled hCG and hLH were equally potent in inhibiting labeled hCG binding, with 50% displacement at 8 ng per tube. Ovine FSH and prolactin did not significantly inhibit binding at doses as high as 10 jug/tube.
Only slight inactivation of [125I]iodo-hCG occurred during the incubation with granulosa cells, based on the capacity of labeled hCG for rebinding to fresh granulosa cells. After incubation for 0.5, 1, and 2h at 37 C, [125I]iodo-hCG retained 96%, 93%, and 90% binding activity compared with the control which was incubated in the buffer without cells, as described in Methods. Quantitative binding
28
1 1
24 20 16 12 8
2 4 6 8 Number of cells (10~6/tube)
10
FIG. 2. Effect of number of cells on [125I]iodohCG binding to medium follicle cells. Incubation was at 37 C for 1 h.
study
of [}25I]iodo-hCG
Granulosa cells from 3 sizes of follicles were incubated with increasing amounts of [125I]iodo-hCG at 37 C for 5-6 h, at which time binding activity had reached a plateau. The binding of hCG to medium follicle cells as a function of hCG concentrations is shown in Fig. 4. Nonspecific binding was linearly related to the concentration of [125I]iodo-hCG. A saturable specific binding curve was obtained when nonspecific binding was subtracted from total binding (Fig. 4). The Scatchard plot of the specific binding data for the medium follicle cells is illustrated in Fig. 5 (middle curve). The equilibrium dissociation con-
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45
hCG RECEPTORS OF GRANULOSA CELLS
100
80 \ FlG. 3. Displacement of [l25I]iodo-hCG binding to medium follicle cells by unlabeled hormones. Incubation was at 37 C for l h ; [125I]iodo-hCG was 5 ng/tube.
. 60
40
20
1
10'
10z
Unlabeled hormone,
stant, calculated from the Scatchard plot, is 2.1 x 10~10M. The number of binding sites for medium follicle cells was estimated to be 16.9 x 10~15 moles per 10 x 106 cells, which is equivalent to approximately 1,000 hCG molecules per cell. The Scatchard plots of the specific binding data for small follicle cells (left curve) and large follicle cells (right curve) are also illustrated in Fig. 5. It must be noted that 10 x 106 cells/tube were used for both small and medium follicle cells, while only
10 3
10"
ng/tube
2 x 106 cells/tube were used for large follicle cells. The data for dissociation constants and the number of receptor sites per cell for granulosa cells from follicles at various stages of development are compared in Table 1. The values of the dissociation constants for small, medium, and large follicle cells are similar, and average 2.4 x 10~10M. The number of binding sites per cell, however, increases 4-fold from
small to medium follicle cells and 26-fold from small to large follicle cells. Total binding
50
FIG. 4. Effect of [125I]iodohCG concentration on [l25I]iodohCG binding to medium follicle cells. Incubation was at 37 C for 5 h with 10 x 106 cells/ tube.
20
40
[
IZ5
60
80
l] hCG, ng/ml
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Endo • 1976 Vol 99 • No 1
LEE
46
TABLE 1. Dissociation constants (IQi) and numbers of binding sites (n) for the granulosa cells of small, medium, and large follicles Granulosa cells of follicles
M x 10'°
n (molecules hCG/cell)
Small (1-2 mm)
1.6 2.5
300 400
Medium (3-5 mm)
2.1 3.8 2.4
1,000 1,400 1,600
Large (6-12 mm)
2.5 1.7
8,200 10,200
hCG-sensitive adenyl cyclase activity Under the assay conditions (3 mM ATP and 5 mM added MgCl2), the adenyl cyclase activities of granulosa cell homogenates in both the absence and the presence of a saturating concentration of hCG were linearly related to the incubation time up to 20 min at 30 C and were also proportional to the number of cells used, up to 4 x 106 cells/tube (unpublished observations). Figure 6 shows that basal adenyl cyclase activity appeared to be higher in large follicle cells. hCG at a concentration 24
as low as 4 ng/ml was sufficient to cause adenyl cyclase activity of large follicle cells to increase 1.8-fold compared with basal activity. The maximal stimulatory response (13-fold relative to basal activity) of large follicle cells was reached at 1 /ag/ml of hCG (Fig. 6). the apparent concentration which gave half-maximal stimulation (apparent Ks) was 45 ng/ml or 9.8 x 10~10M. The inhibition of cyclase activity at 10 /Ag/ml hCG, shown in Fig. 6, was not frequently observed. Both medium and small follicle cells were much less responsive to hCG stimulation with respect to adenyl cyclase activity. hCG at the maximal stimulating dose (1 /xg/ml) caused only 3.7-fold increase in adenyl cyclase activity of medium follicle cells. The apparent Ks (53 ng/ml) is similar to the value obtained for large follicle cells. Small follicle cells were the least responsive to hCG stimulation. Only a very small increment (1.5-fold) of adenyl cyclase activity was obtained at the concentration of 1 /xg/ ml. The limited response of small follicle cells does not allow an accurate estimation of apparent Ks.
-
FIG. 5. Scatchard plots of [ 125 I]iodo-hCG binding to granulosa cells. Incubation was at 37 C for 5-6 h. (X) small follicle cells, 10 x 106 cells/ tube; (•) medium follicle cells, 10 x 108 cells/tube; (A) large follicle cells, 2 x 106 cells/tube.
8
12
Bound (10"
i5
16
20
24
28
mol/tube)
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hCG RECEPTORS OF GRANULOSA CELLS Discussion In previous reports it was demonstrated that LH receptors in the rat ovary can be induced by pregnant mare's serum gonadotropin and hCG priming. The induced LH receptors in the pseudopregnant rat ovary did not differ from those in the immature ovaiy in terms of affinity and specificity for hLH binding (9). The present study indicates similar phenomena. The increase of hCG receptors in porcine granulosa cells during follicle maturation was not accompanied by a change in binding affinity (or dissociation constant). The same finding was also recently reported for porcine granulosa cells by Kammerman and Ross (7). Soil et al. (15) have demonstrated that the decreased insulin receptors in the liver membrane of the ob/ob mouse are indistinguishable from normal with respect to binding affinity, kinetic association and dissociation, temperature dependence, and biological specificity of the binding reaction. It thus appears that the modulation of target tissue response to polypeptide hormone during various functional states is by means of change in the number of hormone receptor sites and not in the binding affinity. The values of the dissociation constants for hCG binding to small, medium, and large follicle cells are in good agreement with the data reported for the hCG-ovarian receptor interaction (16). The mean value of the dissociation constant (2.4 x 10~ 10 M) for granulosa cells from three sizes of
400
f 200
100
100
1000 10,000
100,000
hCG (ng/ml)
FIG. 6. Dose-response curves for hCG-stimulated adenyl cyclase activity of granulosa cells from large, medium, and small follicles. Cyclase assay was performed as described in the Methods. Vertical line indicates the hCG concentration giving half-maximal stimulation of cyclase activity.
follicles compares favorably with the mean value of 1.7 x 10~10M reported by Kammerman and Ross (7). Since little inactivation of free labeled hCG occurred during incubation with intact cells, an accurate estimation of the dissociation constant can be reasonably achieved without correction for inactivation. The number of hCG receptor sites per cell obtained in this study is also in good agreement with reported values (7). The present study also demonstrates the presence of hCG-sensitive adenyl cyclase in porcine granulosa cells and the close
TABLE 2. Comparison of maximal hCG-stimulated adenyl cyclase activity* and the number of hCG receptor sites of granulosa cells cAMP formed 10~12 mole/20 min
cAMP formed molecules/sec
Number off receptors
cAMP formed molecules/sec
Granulosa cells of follicles
4 x 106 cell
cell
cell
receptor
small medium large
1.9 6.0 55.9
240 750 7,000
350 1,300 9,200
0.69 0.58 0.76
* Adenyl cyclase activity was measured according to the Methods at the maximal stimulating concentration (lMg/ml)ofhCG. f Mean values taken from Table 1.
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48
Endo • 1976 Vol 99 • No 1
LEE
correlation between the number of hCG receptor sites and hCG-stimulated adenyl cyclase activity during granulosa cell differentiation. The increase in hCG receptor sites from small to medium follicle cells and the further increase in receptor sites to large follicle cells was accompanied by a proportional increase in hCGsensitive adenyl cyclase activity. These results are more clearly illustrated in Table 2, in which adenyl cyclase activity was calculated from the maximal stimulatory response of granulosa cells at 1 /ug/ml of hCG. The number of cyclic AMP molecules formed per second per cell is proportional to the number of receptor sites per cell for all granulosa cells. This is indicated by the relatively constant ratio (0.58-0.76) of the number of cyclic AMP molecules formed per molecule of hCG receptor. This result does not imply that there is a proportional relationship between the receptor occupancy and the activity of the catalytic subunit of adenylate cyclase. Table 2 only illustrates that the maximal adenyl cyclase activated at the particular hCG concentration studied (1 jug/ml) is dependent on the number of hCG receptor sites available. Enhanced activity of catalytic subunits of the adenyl cyclase of granulosa cells during follicle maturation probably does not occur, as evidenced by the observations that there was no difference in adenyl cyclase activation by NaF and by Prostaglandins Ej and E2 among small, medium, and large follicle cells (unpublished data). Channing and Kammerman (5) have reported an increase in the hCG-binding activity of porcine granulosa cells during follicle development. The enhanced binding activity of large follicle cells was shown to correlate with increases in cyclic AMP production and progesterone synthesis (8). Scanning electron microscopy studies revealed unique changes of the plasma membrane surface of granulosa cells during
follicle development (Chang, S. C., W. Anderson, C. Y. Lee, and R. J. Ryan, manuscript in preparation). Massive numbers of microvilli appeared on large follicle cell surfaces, in contrast to the relatively smooth cell surfaces of small follicle cells. Although the size of granulosa cells from all sizes of follicles has been reported to remain constant (5), the increased number of binding sites per cell does not necessarily imply an increase in binding sites per unit of surface area, since cell surface area may increase without change in cell size. Whether the newly developed hCG receptor sites of large follicle cells are on the microvilli needs to be determined. References 1. Lee, C. Y., and R. J. Ryan, Endocrinology 89: 1515, 1971. 2. Danzo, B. J., A. R. Midgley, and L. J. Kleinsmith, Proc Soc Exp Biol Med 129: 88, 1972. 3. Catt, K. J., M. L. Dufau, and T. Tsuruhara, J Clin Endocrinol Metab 37: 860, 1971. 4. Kammerman, S., R. E. Canfield, J. Kolena, and C. P. Channing, Endocrinology 91: 65, 1972. 5. Channing, C. P., and S. Kammerman, Endocrinology 92: 531, 1973. 6. Lee, C. Y., In Vitro 10: 343, 1974 (Abstract). 7. Kammerman, S., and J. Ross, J Clin Endocrinol Metab 41: 546, 1975. 8. Channing, C. P., and F. Ledwitz-Rigby, In Hardman, J. G., and B. W. O'Malley (eds.), Methods of Enzymology, vol. 39, Academic Press, New York, 1975, p. 183. 9. Lee, C. Y., and R. J. Ryan, Biochemistry 12: 4609, 1973. 10. Ryan, R. J. J Clin Endocrinol Metab 28: 886,1968. 11. Birnbaumer, L., and Po-Chang Yang, J Biol Chem 249: 7867, 1974. 12. Hunzicker-Dunn, M., and L. Birnbaumer, Endocrinology (In press). 13. Salomon, Y., C. Londos, and M. Rodbell, Anal Biochem 58: 541, 1974. 14. Lowry, O. H., N. Y. Rosebrough, A. L. Farr, and R. J. Randall J Biol Chem 193: 265, 1951. 15. Soil, A. H., and C. R. Kahn, J Biol Chem 250: 4702, 1975. 16. Lee, C. Y., and R. J. Ryan, In Endocrine Function of the Human Ovary, Serono Symposium, Florence, 1975 (In press).
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