0021-972X/78/4703-0529$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 47, No. 3 Printed in U.S.A.
Studies of the Human Testis. X. Properties of Human Chorionic Gonadotropin Receptor in Adult Testis and Relation to Intratesticular Testosterone Concentration AN-FEI HSU, DEBORAH STRATICO, MASHIKO HOSAKA, AND PHILIP TROEN Department of Medicine, Montefiore. Hospital, and the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 ABSTRACT. Receptors for [125I]hCG were found in adult human testis. The specific binding of [125I]hCG to testicular receptor is temperature dependent and is a saturable process with respect to added receptor protein and hormone. Scatchard analysis revealed a dissociation constant of 5.0 x 10~10 M, and 6.2 fmol binding site/mg protein. Intact unlabeled hCG effectively inhibits the specific binding of [l25I]hCG to human testicular receptors. For inhibition of binding of [125I]hCG, the a subunit has 3.0% of the potency of intact hCG and the /? subunit has 0.04% of the potency of intact hCG. Specific binding
A
NUMBER of studies have reported the specific uptake of radioiodinated hCG by rat testis (1-3), bovine corpora (4, 5), ovary (6), isolated granulosa cells (7), adrenocortical carcinoma (8), and, most recently, human fetal testis (9, 10). The hCG receptors have been shown to be functionally coupled to adenylate cyclase and testosterone synthesis in intact rat Leydig cells (11, 12). Recently, receptors have been solubilized by nonionic detergents, such as Triton X-100 and Lubrol (13), and also partly purified and characterized (13, 14). However, little information is available on the biochemical properties of the testicular receptor for hCG in human testis. The present studies were performed to determine the presence and properties of an hCG receptor in the adult human testis. In addition, the binding of hCG to human testis has been correlated with the intratesticular concentration of testosterone.
is pH dependent, with an optimum at pH 7.4. Brief exposure to extremes of pH causes irreversible damage to the receptors. Incubation with protease and trypsin results in an almost complete loss of binding activity, while ribonuclease, deoxyribonuclease, phospholipase C, or neuraminidase treatment does not significantly alter hormone-binding activity. Binding activity was found to be positively correlated to the concentration of intratesticular testosterone. (JClin Endocrinol Metab 47: 529, 1978)
and 5.0 IU/mg, respectively) were a gift from the Center for Population Research, NICHHD, NIH. Other chemicals used and vendors are as follows: carrier-free [125I]NaI and [l,2-3H]testosterone (SA, 50 Ci/mmol) were obtained from the New England Nuclear Corp.; deoxyribonuclease I and ribonuclease A were obtained from Sigma Chemical Co.; trypsin, protease, neuraminidase, and phospholipase C were purchased from Worthington Biochemical Corp.; unlabeled hCG for nonspecific binding was purchased from Ayerst Laboratories. Preparation of [125IJhCG Unlabeled hCG from NIH was iodinated using the chloramine-T method, as described by Greenwood et al. (15). The labeled hormone was further purified by Sepharose-Concanavalin A (16). Iodinated hCG was kept frozen in small aliquots until used, for periods of less than 4 weeks. Before use it was diluted in phosphate-buffered saline (PBS) containing 0.1% y-globulin. Purification of unlabeled hCG for nonspecific binding
Materials and Methods Unlabeled hCG for iodination (biological potency, 11,600 IU/mg) and its a and /? subunits (14.5 Received September 14,1977. Address requests for reprints to: Dr. Philip Troen, Montefiore Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213.
Unlabeled hCG from a commercial source was purified on a 2.5 X 40-cm Sephadex G-100 column eluting with 0.05 M sodium phosphate (pH 7.4). The protein profile was obtained by reading the optical density at 280 nm. The protein peak corresponding to [125I]hCG was collected and concentrated by
529
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530
JCE&M VoU7
HSU ET AL.
pressure dialysis. The protein concentration was determined by Fluram assay (17). An RIA (18) was performed to determine the concentration of hCG in the purified protein, using unlabeled hCG from NIH as a standard. Anti-hCG was generated in female New Zealand rabbits using conventional immunization procedures (19). Preparation of testicular fraction for receptor studies Human testicular tissue was obtained from patients (75-90 yr of age) undergoing orchiectomy for prostatic cancer. None of the patients had received prior hormone therapy. In one instance, orchiectomy was performed on a 21-yr-old patient at the time of surgery for cryptorchidism. The fresh tissue was homogenized in PBS using a motor-driven glass-Teflon Potter-Elvehjem homogenizer, then passed through four layers of cheesecloth and centrifuged at 3500 rpm for 15 min. The pellet was dissolved in PBS and the protein concentration was determined by the method of Lowry et al. (20). This testicular preparation was used for the testicular receptor-binding studies in this paper.
1978 No 3
value, which ranged from 20-30 ju,Ci/ju.g, was used to calculate the total concentration of intact [125I]hCG. The concentration of bound hormone was calculated after correction for maximum bindability of labeled hormone to receptor. Maximum bindability ranged from 40-50% in our experiments. Free bindable hormone concentration was derived by subtracting bound from total hormone concentrations. The molar concentrations of hCG were calculated using a correction of the particular hCG preparation used to the expected potency of 15,000 IU/mg for fully active hCG. Determination of intratesticular testosterone concentration
Testicular tissue (2-5 mg) obtained at orchiectomy, as indicated above, was homogenized in 1.0 ml ice-cold 0.25 M sucrose solution (pH 7.4). [1,23 H]Testosterone (3000 dpm) was added to tubes containing homogenate equivalent to 0.4 mg testicular tissue to allow for correction for loss of endogenous testosterone during the RIA. The homogenates were then extracted with 2.0 ml distilled ether and centrifuged at 3000 rpm for 10 min. The ether phase was removed and the extraction was reSeparation of bound and free hormone peated. Redistilled water (0.5 ml) was added to wash the combined ether extracts. The ether phase Aliquots of the above testicular preparation conwas then dried under vacuum. The sample was taining known amounts of protein (2-3 mg) were loaded on a Sephadex LH-20 microcolumn with the added to the 13 X 75-mm disposable polyethylene solvent system hexane-benzene-methanol (80:15:5, 125 tubes containing [ I]hCG (50-100,000 cpm) and vol/vol) to separate testosterone from other steincubated in triplicate in a Dubnoff metabolic roids. Recovery of initial radioactivity was detershaker. PBS containing 0.1% y-globulin was added mined by aliquoting half of the eluates at chromato make the final incubation volume 0.5 ml. Nontography into a scintillation vial for counting in a specific binding was determined in each experiment Packard Tri-Carb scintillation counter. The ex125 by incubation with [ I]hCG and a 1000-fold excess tracted purified testosterone was measured in the unlabeled hCG. After incubation, 1.0 ml ice-cold other half of the eluates by specific RIA (18). The PBS was added to each tube. The tubes were antiserum has 7% cross-reactivity with 5a-dihydrovortexed and centrifuged immediately at 3500 rpm testosterone. The latter as well as other steroids for 15 min. The supernatants were discarded and were separated from testosterone by the Sephadex the pellets were washed and centrifuged two addicolumn. The blank values in tissue previously extional times to remove excess counts. The pellets tracted with ether were 24.4 ± 17.4 (SD) pg/sample. were then counted in a Nuclear Chicago (Searle) Water was used for a blank control. The interassay autogamma counter. Nonspecific binding was subcoefficient of variation was 8.3% and the intraassay tracted from total binding to obtain specific bindcoefficient of variation was 9.4%. ing. Data are presented as specific binding.
Results Calculation of the total, bound, and free hormone Effects of time and temperature on the assoconcentrations 125
ciation of
I-labeled hCG to human testicu-
This was carried out as described by Ketelslegers lar receptor et al. in 1975 (21). The specific activity of the The specific binding of 125I-labeled hCG to [I25I]hCG preparation was determined by self-displacement assay of increasing concentrations of human testicular receptor is dependent on tracer mass in a radioligand receptor assay. This both the time and temperature of incubation,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 03:09 For personal use only. No other uses without permission. . All rights reserved.
hCG RECEPTOR AND TESTOSTERONE IN HUMAN TESTIS
531
not studied. A study of the effect of calcium on [125I]hCG and testicular receptors was carried out in the presence of zero to three times physiological levels of calcium ions. No differences in the binding were detectable.
as shown in Fig. 1. Uptake of labeled hCG was higher at 27 C than at 37 C, but both reached a maximum at 8 h. When incubation was continued overnight at 37 C, the binding declined more than at 27 C, possibly due to more rapid degradation of the labeled hormone during incubation at 37 C. The rate of association at 4 C was extremely slow; no significant amount of binding was obtained even after 24 h of incubation. The effect of temperature and length of incubation on the dissociation of [125I]hCG from human testicular receptor was
Effect of increasing concentrations of protein on the binding of [125IJhCG The extent of binding of [125I]hCG to human testicular preparation is proportional to the protein concentration (Fig. 2), reaching maxi-
b
2
4
INCUBATION
TIME (hours)
FIG. 1. Time course study for specific binding of ['"f>I]hCG to a human testicular preparation at various temperatures. Protein (2.0 mg) was incubated with [l2r'I]hCG (50-100,000 cpm) at 4, 27, and 37 C for different lengths of time.
Z. 1000 -
o
FIG. 2. Specific binding of [125I] hCG to a human testicular preparation with increasing concentrations of protein. [125I]hCG (50-100,000 cpm) was incubated at 37 C for 8 h with increasing concentrations of protein (1-5 mg).
500 -
2 PROTEIN
3
4
CONCENTRATION
(mg)
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J C E & M • 1978 Vol 47 • No 3
HSU ET AL.
532
mum binding at 5 mg protein. In the present studies, 1.5-2 mg protein/tube were usually used for incubations, and specific binding of biologically active 12r>I-labeled hCG was generally from 4-12%/incubation. Nonspecific binding varied from 10-30% of specific binding. Extent of affinity of specific hCG binding As shown in Fig. 3A, the specific binding of [125I]hCG to receptors in testis is a saturable process. The relation between gonadotropin uptake by testicular preparation and concentration of gonadotropin was determined by measurement of [125I]hCG binding in the presence of increasing quantities of labeled gonadotropin. Unlabeled hCG was increased proportionally to measure nonspecific binding. Specific binding of [125I]hCG increased with the amount of labeled hormone added, and reached a plateau at low concentration of hormone (3-4 ng hCG). Figure 3B depicts the Scatchard plot (22) analysis of the data in Fig. 3A; the estimate of equilibrium association constant (Ka) was 0.20 X 1010 M~\ and the binding capacity from the intercept on the abscissa gave 6.2 fmol/mg protein (62 fmol/g testis) as the maximum amount of hCG bound. The reciprocal of the slope yields the dissociation constant (Kd), which is 5.0 x 10"10 M. m
Displacement of [ IJhCG binding by unlabeled hCG and hCG subunits The presence of increasing quantities of unlabeled hCG and hCG-a and hCG-/? subunits caused progressive displacement of [125I]hCG binding from human testicular receptor (Fig. 4). Almost total inhibition of the binding of labeled hCG (0.29 ng) was obtained with 0.2-0.3 jiig unlabeled hCG. Displacement of [125I]hCG binding by hCG-a subunit required a 30- to 40-fold greater concentration of a subunit than the concentration of intact hCG to produce a similar inhibition; displacement by hCG-/? subunit needed at least 2000 times the concentration of intact hCG. Some of the displacement found with high concentration of subunits may be due to contamination with intact hCG.
Effect ofpH Binding of [125I]hCG to human testicular receptor occurs over a fairly narrow range of pH. As shown in Fig. 5, maximal binding occurred at pH 7.4. It is worth noting that the receptor retained less than 50% of its binding activity at pH above 8.5 or below 6. The binding activity of human testicular receptors was destroyed irreversibly by a brief exposure to pH above 10 or below 5. Effect of enzyme treatment on testicular receptor-binding activity The chemical nature of receptor was investigated by treating the receptor protein fraction with different enzymes, followed by testing for specific binding to [125I]hCG. As shown in Table 1, proteolytic enzymes, such as protease and trypsiri;* abolished over 80% of the binding activity of the receptor, while ribonuclease and deoxyribonuclease treatments did not affect the receptor binding. Treatment with neuraminidase and phospholipase C also did not significantly alter the binding activity of the receptor. Relationship between [125IJhCG binding to human testicular receptors and intratesticular concentrations of testosterone The concentration of intratesticular testosterone ranged from 437-1100 ng/g tissue in the six patients studied. Corresponding values or the percentage of specific hCG binding per mg protein ranged from 0.45-7.12%. As shown in Fig. 6, there is a high positive linear correlation (r = 0.97) between specific hCG binding and intratesticular testosterone concentration. Discussion This study demonstrates the binding of [125I]hCG by adult human testis. The receptor is characterized by high affinity and low saturation. Binding is temperature and time dependent and is readily inhibited by intact unlabeled hCG but not by the hCG subunits. Binding capacity is lost after treatment with protease and trypsin. The characteristics of a
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 03:09 For personal use only. No other uses without permission. . All rights reserved.
hCG RECEPTOR AND TESTOSTERONE IN HUMAN TESTIS
a.
2
4
FIG. 3. A, Specific binding (• • ) of [125I]hCG to a human testicular preparation with increasing concentrations of [125I] hCG. Protein (2.0 mg) was incubated at 37 C for 7 h at indicated concentrations of [125I]hCG. Total binding (O O) and nonspecific binding (x X) are indicated. B, Scatchard plot of [125I]hCGbinding data from A. The K
Vol. 47, No. 3 Printed in U.S.A.
Studies of the Human Testis. X. Properties of Human Chorionic Gonadotropin Receptor in Adult Testis and Relation to Intratesticular Testosterone Concentration AN-FEI HSU, DEBORAH STRATICO, MASHIKO HOSAKA, AND PHILIP TROEN Department of Medicine, Montefiore. Hospital, and the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213 ABSTRACT. Receptors for [125I]hCG were found in adult human testis. The specific binding of [125I]hCG to testicular receptor is temperature dependent and is a saturable process with respect to added receptor protein and hormone. Scatchard analysis revealed a dissociation constant of 5.0 x 10~10 M, and 6.2 fmol binding site/mg protein. Intact unlabeled hCG effectively inhibits the specific binding of [l25I]hCG to human testicular receptors. For inhibition of binding of [125I]hCG, the a subunit has 3.0% of the potency of intact hCG and the /? subunit has 0.04% of the potency of intact hCG. Specific binding
A
NUMBER of studies have reported the specific uptake of radioiodinated hCG by rat testis (1-3), bovine corpora (4, 5), ovary (6), isolated granulosa cells (7), adrenocortical carcinoma (8), and, most recently, human fetal testis (9, 10). The hCG receptors have been shown to be functionally coupled to adenylate cyclase and testosterone synthesis in intact rat Leydig cells (11, 12). Recently, receptors have been solubilized by nonionic detergents, such as Triton X-100 and Lubrol (13), and also partly purified and characterized (13, 14). However, little information is available on the biochemical properties of the testicular receptor for hCG in human testis. The present studies were performed to determine the presence and properties of an hCG receptor in the adult human testis. In addition, the binding of hCG to human testis has been correlated with the intratesticular concentration of testosterone.
is pH dependent, with an optimum at pH 7.4. Brief exposure to extremes of pH causes irreversible damage to the receptors. Incubation with protease and trypsin results in an almost complete loss of binding activity, while ribonuclease, deoxyribonuclease, phospholipase C, or neuraminidase treatment does not significantly alter hormone-binding activity. Binding activity was found to be positively correlated to the concentration of intratesticular testosterone. (JClin Endocrinol Metab 47: 529, 1978)
and 5.0 IU/mg, respectively) were a gift from the Center for Population Research, NICHHD, NIH. Other chemicals used and vendors are as follows: carrier-free [125I]NaI and [l,2-3H]testosterone (SA, 50 Ci/mmol) were obtained from the New England Nuclear Corp.; deoxyribonuclease I and ribonuclease A were obtained from Sigma Chemical Co.; trypsin, protease, neuraminidase, and phospholipase C were purchased from Worthington Biochemical Corp.; unlabeled hCG for nonspecific binding was purchased from Ayerst Laboratories. Preparation of [125IJhCG Unlabeled hCG from NIH was iodinated using the chloramine-T method, as described by Greenwood et al. (15). The labeled hormone was further purified by Sepharose-Concanavalin A (16). Iodinated hCG was kept frozen in small aliquots until used, for periods of less than 4 weeks. Before use it was diluted in phosphate-buffered saline (PBS) containing 0.1% y-globulin. Purification of unlabeled hCG for nonspecific binding
Materials and Methods Unlabeled hCG for iodination (biological potency, 11,600 IU/mg) and its a and /? subunits (14.5 Received September 14,1977. Address requests for reprints to: Dr. Philip Troen, Montefiore Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213.
Unlabeled hCG from a commercial source was purified on a 2.5 X 40-cm Sephadex G-100 column eluting with 0.05 M sodium phosphate (pH 7.4). The protein profile was obtained by reading the optical density at 280 nm. The protein peak corresponding to [125I]hCG was collected and concentrated by
529
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 03:09 For personal use only. No other uses without permission. . All rights reserved.
530
JCE&M VoU7
HSU ET AL.
pressure dialysis. The protein concentration was determined by Fluram assay (17). An RIA (18) was performed to determine the concentration of hCG in the purified protein, using unlabeled hCG from NIH as a standard. Anti-hCG was generated in female New Zealand rabbits using conventional immunization procedures (19). Preparation of testicular fraction for receptor studies Human testicular tissue was obtained from patients (75-90 yr of age) undergoing orchiectomy for prostatic cancer. None of the patients had received prior hormone therapy. In one instance, orchiectomy was performed on a 21-yr-old patient at the time of surgery for cryptorchidism. The fresh tissue was homogenized in PBS using a motor-driven glass-Teflon Potter-Elvehjem homogenizer, then passed through four layers of cheesecloth and centrifuged at 3500 rpm for 15 min. The pellet was dissolved in PBS and the protein concentration was determined by the method of Lowry et al. (20). This testicular preparation was used for the testicular receptor-binding studies in this paper.
1978 No 3
value, which ranged from 20-30 ju,Ci/ju.g, was used to calculate the total concentration of intact [125I]hCG. The concentration of bound hormone was calculated after correction for maximum bindability of labeled hormone to receptor. Maximum bindability ranged from 40-50% in our experiments. Free bindable hormone concentration was derived by subtracting bound from total hormone concentrations. The molar concentrations of hCG were calculated using a correction of the particular hCG preparation used to the expected potency of 15,000 IU/mg for fully active hCG. Determination of intratesticular testosterone concentration
Testicular tissue (2-5 mg) obtained at orchiectomy, as indicated above, was homogenized in 1.0 ml ice-cold 0.25 M sucrose solution (pH 7.4). [1,23 H]Testosterone (3000 dpm) was added to tubes containing homogenate equivalent to 0.4 mg testicular tissue to allow for correction for loss of endogenous testosterone during the RIA. The homogenates were then extracted with 2.0 ml distilled ether and centrifuged at 3000 rpm for 10 min. The ether phase was removed and the extraction was reSeparation of bound and free hormone peated. Redistilled water (0.5 ml) was added to wash the combined ether extracts. The ether phase Aliquots of the above testicular preparation conwas then dried under vacuum. The sample was taining known amounts of protein (2-3 mg) were loaded on a Sephadex LH-20 microcolumn with the added to the 13 X 75-mm disposable polyethylene solvent system hexane-benzene-methanol (80:15:5, 125 tubes containing [ I]hCG (50-100,000 cpm) and vol/vol) to separate testosterone from other steincubated in triplicate in a Dubnoff metabolic roids. Recovery of initial radioactivity was detershaker. PBS containing 0.1% y-globulin was added mined by aliquoting half of the eluates at chromato make the final incubation volume 0.5 ml. Nontography into a scintillation vial for counting in a specific binding was determined in each experiment Packard Tri-Carb scintillation counter. The ex125 by incubation with [ I]hCG and a 1000-fold excess tracted purified testosterone was measured in the unlabeled hCG. After incubation, 1.0 ml ice-cold other half of the eluates by specific RIA (18). The PBS was added to each tube. The tubes were antiserum has 7% cross-reactivity with 5a-dihydrovortexed and centrifuged immediately at 3500 rpm testosterone. The latter as well as other steroids for 15 min. The supernatants were discarded and were separated from testosterone by the Sephadex the pellets were washed and centrifuged two addicolumn. The blank values in tissue previously extional times to remove excess counts. The pellets tracted with ether were 24.4 ± 17.4 (SD) pg/sample. were then counted in a Nuclear Chicago (Searle) Water was used for a blank control. The interassay autogamma counter. Nonspecific binding was subcoefficient of variation was 8.3% and the intraassay tracted from total binding to obtain specific bindcoefficient of variation was 9.4%. ing. Data are presented as specific binding.
Results Calculation of the total, bound, and free hormone Effects of time and temperature on the assoconcentrations 125
ciation of
I-labeled hCG to human testicu-
This was carried out as described by Ketelslegers lar receptor et al. in 1975 (21). The specific activity of the The specific binding of 125I-labeled hCG to [I25I]hCG preparation was determined by self-displacement assay of increasing concentrations of human testicular receptor is dependent on tracer mass in a radioligand receptor assay. This both the time and temperature of incubation,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 03:09 For personal use only. No other uses without permission. . All rights reserved.
hCG RECEPTOR AND TESTOSTERONE IN HUMAN TESTIS
531
not studied. A study of the effect of calcium on [125I]hCG and testicular receptors was carried out in the presence of zero to three times physiological levels of calcium ions. No differences in the binding were detectable.
as shown in Fig. 1. Uptake of labeled hCG was higher at 27 C than at 37 C, but both reached a maximum at 8 h. When incubation was continued overnight at 37 C, the binding declined more than at 27 C, possibly due to more rapid degradation of the labeled hormone during incubation at 37 C. The rate of association at 4 C was extremely slow; no significant amount of binding was obtained even after 24 h of incubation. The effect of temperature and length of incubation on the dissociation of [125I]hCG from human testicular receptor was
Effect of increasing concentrations of protein on the binding of [125IJhCG The extent of binding of [125I]hCG to human testicular preparation is proportional to the protein concentration (Fig. 2), reaching maxi-
b
2
4
INCUBATION
TIME (hours)
FIG. 1. Time course study for specific binding of ['"f>I]hCG to a human testicular preparation at various temperatures. Protein (2.0 mg) was incubated with [l2r'I]hCG (50-100,000 cpm) at 4, 27, and 37 C for different lengths of time.
Z. 1000 -
o
FIG. 2. Specific binding of [125I] hCG to a human testicular preparation with increasing concentrations of protein. [125I]hCG (50-100,000 cpm) was incubated at 37 C for 8 h with increasing concentrations of protein (1-5 mg).
500 -
2 PROTEIN
3
4
CONCENTRATION
(mg)
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J C E & M • 1978 Vol 47 • No 3
HSU ET AL.
532
mum binding at 5 mg protein. In the present studies, 1.5-2 mg protein/tube were usually used for incubations, and specific binding of biologically active 12r>I-labeled hCG was generally from 4-12%/incubation. Nonspecific binding varied from 10-30% of specific binding. Extent of affinity of specific hCG binding As shown in Fig. 3A, the specific binding of [125I]hCG to receptors in testis is a saturable process. The relation between gonadotropin uptake by testicular preparation and concentration of gonadotropin was determined by measurement of [125I]hCG binding in the presence of increasing quantities of labeled gonadotropin. Unlabeled hCG was increased proportionally to measure nonspecific binding. Specific binding of [125I]hCG increased with the amount of labeled hormone added, and reached a plateau at low concentration of hormone (3-4 ng hCG). Figure 3B depicts the Scatchard plot (22) analysis of the data in Fig. 3A; the estimate of equilibrium association constant (Ka) was 0.20 X 1010 M~\ and the binding capacity from the intercept on the abscissa gave 6.2 fmol/mg protein (62 fmol/g testis) as the maximum amount of hCG bound. The reciprocal of the slope yields the dissociation constant (Kd), which is 5.0 x 10"10 M. m
Displacement of [ IJhCG binding by unlabeled hCG and hCG subunits The presence of increasing quantities of unlabeled hCG and hCG-a and hCG-/? subunits caused progressive displacement of [125I]hCG binding from human testicular receptor (Fig. 4). Almost total inhibition of the binding of labeled hCG (0.29 ng) was obtained with 0.2-0.3 jiig unlabeled hCG. Displacement of [125I]hCG binding by hCG-a subunit required a 30- to 40-fold greater concentration of a subunit than the concentration of intact hCG to produce a similar inhibition; displacement by hCG-/? subunit needed at least 2000 times the concentration of intact hCG. Some of the displacement found with high concentration of subunits may be due to contamination with intact hCG.
Effect ofpH Binding of [125I]hCG to human testicular receptor occurs over a fairly narrow range of pH. As shown in Fig. 5, maximal binding occurred at pH 7.4. It is worth noting that the receptor retained less than 50% of its binding activity at pH above 8.5 or below 6. The binding activity of human testicular receptors was destroyed irreversibly by a brief exposure to pH above 10 or below 5. Effect of enzyme treatment on testicular receptor-binding activity The chemical nature of receptor was investigated by treating the receptor protein fraction with different enzymes, followed by testing for specific binding to [125I]hCG. As shown in Table 1, proteolytic enzymes, such as protease and trypsiri;* abolished over 80% of the binding activity of the receptor, while ribonuclease and deoxyribonuclease treatments did not affect the receptor binding. Treatment with neuraminidase and phospholipase C also did not significantly alter the binding activity of the receptor. Relationship between [125IJhCG binding to human testicular receptors and intratesticular concentrations of testosterone The concentration of intratesticular testosterone ranged from 437-1100 ng/g tissue in the six patients studied. Corresponding values or the percentage of specific hCG binding per mg protein ranged from 0.45-7.12%. As shown in Fig. 6, there is a high positive linear correlation (r = 0.97) between specific hCG binding and intratesticular testosterone concentration. Discussion This study demonstrates the binding of [125I]hCG by adult human testis. The receptor is characterized by high affinity and low saturation. Binding is temperature and time dependent and is readily inhibited by intact unlabeled hCG but not by the hCG subunits. Binding capacity is lost after treatment with protease and trypsin. The characteristics of a
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 03:09 For personal use only. No other uses without permission. . All rights reserved.
hCG RECEPTOR AND TESTOSTERONE IN HUMAN TESTIS
a.
2
4
FIG. 3. A, Specific binding (• • ) of [125I]hCG to a human testicular preparation with increasing concentrations of [125I] hCG. Protein (2.0 mg) was incubated at 37 C for 7 h at indicated concentrations of [125I]hCG. Total binding (O O) and nonspecific binding (x X) are indicated. B, Scatchard plot of [125I]hCGbinding data from A. The K
