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Binding of follicle-stimulating hormone by homogenates of testes of photostimulated white-crowned sparrows, Zonotrichia leucophrys gambelii
GENERAL
AND
COMPARATIVE
Binding
ENDOCRINOLOGY
30, 443-450 (1976)
of Follicle-Stimulating Hormone by Homogenates of Testes of Photostimulated White-Crowned Sparrows, Zonotrichia leucophrys gambelii SUSUMU ISHII AND DONALD S. FARNER Department of Biology, School of Education, Waseda University, Tokyo 160, Japan, and Department of Zoology, University of Washington, Seattle, Washington 98195
Accepted July 16, 1976 Homogenates of testes of the white-crowned sparrow bind specifically with 1251-labeled rat FSH (follicle-stimulating hormone). The binding is competitively inhibited by unlabeled rat FSH and PMSG (pregnant mare serum gonadotropin) but not by purified rat LH (luteinizing hormone). Crude gonadotropin preparations also inhibit binding but the rates of inhibition are fully explained by amounts of FSH in these preparations. The testes of photosensitive birds with resting testes were found to bind about 4 x lo-i5 moles of ‘“I-labeled rat FSH per milligram of tissue. The binding per milligram of tissue decreases during photoperiodically induced testicular growth, although the total binding capacity increases during the first 3 weeks of photoperiodic stimulation. Thereafter it increases no further although the weight of the testes continues to increase rapidly. With further development, a specific and sensitive radioreceptor assay of avian FSH, using a testicular homogenate of the white-crowned sparrow as the receptor, may be possible.
It has been established in mammals that gonadotropins, as well as the other peptide hormones, exert their action by binding to specific receptors on the membrane of the target cells in gonads (e.g., Catt and Dufau, 1973; Lee and Ryan, 1973; Means, 1973). Accordingly, considerations of hormonal control of the gonad must take into account not only plasma levels of the hormones but also the binding of the hormones to the specific receptors in the target organ. The testes of photoperiodic birds in the photosensitive state undergo rapid growth under long-day conditions. In the white-crowned sparrow they attain a weight that is about 200 times the initial weight during 5 weeks of the photostimulation with 16 hr of light per day, 16L 8D (Follett et al., 1975). A remarkable increase in plasma gonadotropin during photostimulation has been reported in this species (Follett et al., 1975) and in the Japanese quail (e.g., Follett,
1976). However, to the best of our knowledge, no investigation has been published on the change in the gonadotropin binding by the avian gonad during photostimulation or even on the gonadotropin binding per se by the avian gonad. In this communication, we report that the testis of the white-crowned sparrow has specific binding sites for follicle stimulating hormone (FSH) and also that the binding capacity changes with the development of the testis under artificial photostimulation. MATERIALS
443 Copyright All rights
@ 1976 by Academic Press, inc. of reproduction in any form reserved.
AND METHODS
Adult male white-crowned sparrows were obtained with mist nets from migrating or wintering populations near Ellensburg, Kittitas County, and on the Sunnyside Game Refuge in Yakima County, both in central Washington, during the autumn of 1974 and late winter of 1975. After temporary confinement in large outdoor aviaries under natural conditions in Seattle, 38 were transferred to a constant-condition chamber (20 ? O.S”C, R.H. ca. 45%, 8-hr daily
444
ISHII
AND
photophase. 8L 16D) more than 6 months before they were used for the experiments described herein. They were divided at random into six groups: Group 1 (14 birds), Group II (8 birds), Groups III-VI (4 each): one bird was lost from Group III. Groups II-VI were exposed to 20-hr daily photophases (20L 4D) for 1, 2, 3, 4, and 5 weeks, respectively. At the end of the longday treatment, the birds were sacrificed for determination of the binding of FSH by the testes. Ten birds (Group VII), which had been captured in October 1975 and held in an outdoor aviary until 1 December, were transferred directly into a long-day chamber and were photostimulated for 8 days. All birds of Groups I-VII were sacrificed between 8 and 10 December, according to the schedule in Table 1. Binding experiments. After sacrifice by decapitation, testes of the birds were removed and weighed. All of the following procedures were performed under chilled conditions unless specified. Testes were homogenized (10 mg of tissue and 1 ml of medium) with a glass homogenizer with a Teflon rod by moving the rod up and down 10 times. The medium, Tris buffer-MgCl,BSA, consisted of 40 mM Tris-HCI buffer (pH 7.5) made 5 mM with MgCI, and 0.1% with bovine serum albumin. The testes of the birds in Groups I, II, and VII, respectively, were pooled for the homogenization. In Group III, testes of two or three birds were combined and homogenized. In Groups IV-VI, testes of each bird were homogenized separately. Homogenates were filtered through a layer of cheesecloth to remove fragments of tunica albuginea. Aliquots of the homogenates were then centrifuged at 5000 rpm in a refrigerated centrifuge for 10 min. The supernatant fluids were decanted and the sediments were resuspended with the buffer solution. The final concentration of the resuspended homogenate was adjusted to 2.5 mg of tissue/O.1 ml in ah experiments except the one that
FARNER
required a series of concentrations of tissue. Then 0.1 ml of homogenate, 20 ~1 of a radioiodinated rat follicle-stimulating hormone (i251-FSH) solution and 20 ~1 of a cold rat FSH solution, or the vehicle alone, were mixed in a glass tube coated with silicone and incubated at 37” with continuous shaking. The incubation time was 5 hr in all of the experiments except the one in which incubation time was varied. The radioiodinated hormone and cold hormone were dissolved in 10 mM phosphate buffer made up to 0.15 M with NaCl and with 0.1% BSA-20 pl of ‘Z51-FSH (13-14 x 104 cpm) and 20 ~1 of cold FSH solution containing 1.2 pg of the cold FSH. In competition experiments different amounts of various gonadotropin preparations were used instead of the cold FSH. At the end of the incubation 1 ml of chilled Tris buffer-MgCl,-BSA was added to each reaction tube, and the reaction tubes were centrifuged at 5000 rpm for 10 min. The sediments thus obtained were washed twice by resuspension and centrifugation. The sediments obtained after the final centrifugation were counted for radioactivity of the lz51-FSH bound to sedimented particles in an automatic gamma counter (Nuclear Chicago). The radioactivity of the sediment of the tube that contained no cold hormone was expressed as the total binding and that of the tube containing 1.2 pg of cold FSH as the nonspecific binding. The difference between the total and nonspecific binding was defined as the specific binding. Because of the limited amount of purified hormones available and the rather low coefficient of variation in the determination of the binding (less than 0.12 in a preliminary experiment), only single determinations were used throughout the experiments. Hormone preparations. NIAMD rat FSH-I-3 was used for radioiodination. Hormone preparations used for the competition experiments were NIAMD rat
TABLE
1
PHOTOSTIMULATION SCHEDULE AND MEAN BODY WEIGHT AT SACRIFICE WHITE-CROWNED SPARROWS IN EACH EXPERIMENTAL GROUP
Group I II III IV V VI VII
Number of birds 14 8 3 4 4 4 10
Photostimulation
(2OL 4D)
Period
Days
I- 8 Dec. 24 Nov.- 8 Dec. 19 Nov.-l0 Dec. 12 Nov.-l0 Dec. 5 Nov.-l0 Dec. I- 9 Dec.
0 7 14 21 28 35 8
a The increase in terminal body weight is the result of photoperiodically of this species.
OF
Terminal mean body weight -c SEM (g)” 26.1 27.1 29.1 29.5 30.0 27.5 28.6
i- 0.7 + 0.7 T’- 1.3 ‘- 1.0 2 2.1 +- 0.7 2 0.8
induced fat deposition characteristic
FSH BINDING
BY
LH-RP-1, NIAMD rat LH-I-4, pregnant mare serum gonadotropin (PMSG) (Sigma), and human chorionic gonadotropin (HCG) (Parke Davis). HCG was first dialyzed against phosphate buffer-saline overnight in order to remove the benzethonium chloride present as a preservative. Iodination procedure. The iodination of purified rat FSH was performed using lactoperoxidase as the catalytic agent. To the reaction tube containing 2.5 Mg of FSH dissolved in 25 ~1 of phosphate buffer-saline were added 30 bl of 0.4 M acetate buffer (pH 5.6), 200 mg of lactoperoxidase (Sigma) dissolved in 5 ~1 of 0.1 M acetate buffer (pH 5.6), 0.5 mCi of carrier-free NaLzSI (Radiochemical Centre, Amersham) dissolved in 5 ~1 of 0.1 N NaOH, and 5 ~1 of a solution containing 75 ng of HZ02. The mixture was held at room temperature for 15 min during which 5 ~1 of the HZOZ solution was added at 5-min intervals (three times in total). The reaction mixture was then transferred to a column of Sephadex G-75. Preparation of the column and separation of ‘*%FSH from inorganic iodide by the column were performed according to the instructions for the radioimmunoassay of rat follicle stimulating hormone, Rat Pituitary Hormone Distribution Program, NIAMDD. The recovery of the hormone in the eluate from the column was estimated according to Greenwood and Hunter (1963). The specific activity of the labeled FSH was estimated to be 45 mCi/mg. Offhand, this activity appears somewhat high, raising the possibility of damage of the hormone. However, comparisons of low- and highactivity preparations on testes from Japanese quail (Ishii, unpublished) revealed no significant differences in results. Furthermore, Bell et al. (1974) have used, with satisfactory results, radioiodinated FSH with a specific activity as high as 60 mCi/mg.
SPARROW
445
TESTES
0
2.5 Tissue
5.0
weight
(me)
FIG. 1. Binding of ‘%FSH to homogenates of the testis of the white-crowned sparrow at varying concentrations of tissue. Each reaction tube contained 13.1 x 104 cpm of *?-FSH; 1.25, 2.5, or 5.0 mg of the testicular tissue; and 1.2 pg of cold FSH or vehicle alone. Incubation was performed at 37” for 3 hr. See text for definitions of total (broken line and solid circles) and specific (continuous line and open circles) bindings.
Total
RESULTS
Binding of lz5Z-FSH as function of incubation time and tissue concentration. Homogenates containing 2.5 mg of testicular tissue from Group I and lz51-FSH were incubated with or without an excess amount of cold FSH for 1, 2, and 3 hr, and the total and nonspecific bindings were measured (Figs. 1 and 2). Both types of binding proceeded rapidly for about 1 hr and then increased gradually for the next 2 hr (Fig. 2), reaching an equilibrium in approximately 3 hr. The ratio of specific to total binding at equilibrium was 53%. Homogenates containing 2.5, 5.0, and 10.0 mg of tissue from Group I were incubated with 1251-FSH and an excess amount
Incubation
time
(hr)
2. Binding of ‘*%I-FSH to homogenates of the testis of the white-crowned sparrow at different times of incubation. Each reaction tube contained 13.1 x 104 cpm of Y-FSH, 2.5 mg of the tissue, and 1.2 /Ig of cold FSH or vehicle alone. Incubation was performed at 37” for 1, 2, or 3 hr. See text for definitions of total (broken line and solid circles) and specific (continuous line and open circles) bindings. FIG.
446
ISHII AND FARNER
of cold FSH or with ‘“51-FSH alone for 3 hr, and the total and nonspecific bindings were measured. Both total and specific bindings were nonlinear as a function of the concentration of the homogenate under the conditions used in this experiment (Fig. 1). The ratio of specific to total binding at equilibrium was 53%. Competition of binding of ‘251-FSH by various gonadotropin preparations. Homogenates of testicular tissue derived from Group VII were incubated with 1251FSH with increasing amounts of the various gonadotropin preparations. NIAMD rat FSH-I-3, the preparation used for the labeled hormone, competed with the binding of the labeled hormone in small amounts (Fig. 3); 50% competition was obtained at about 3 ng. NIAMD rat FSH-RP1, NIAMD rat LH-RP-1, and PMSG also showed clear competition at moderate amounts (Fig. 3). The slopes of the competition curves were similar among these hormone preparations. NIAMD rat LH-I-4 showed essentially no competition even when present in large amounts, ca. 2 pg. HCG competed only at extremely large amounts, ca. 1 mg (Fig. 3). The relative FSH potencies of the NIAMD gonadotropin preparations and
d
HCG were calculated from distances on the competition curves at about the 50% competition level and are given in Table 2. The statistical analyses of the competition curves in Fig. 3 are given in Table 3. The curves can be regarded as parallel. The relative FSH potencies thus obtained coincide with FSH potencies determined by bioassay, as shown in the instructions from NIAMDD. A Scatchard plot of the data from the competition experiment with NIAMD-rat FSH-I-3 was performed after removing the two marginal values. The dissociation constant at equilibrium and the capacity for binding obtained by the Scatchard plot were 0.78 nM and 7.0 fmoles of FSH/mg of tissue, respectively. Change in the binding with development of testes. The weight of the testes of the photostimulated birds continued to increase through the stimulation period and became maximal at the fifth week (Fig. 4). The increase was especially marked between 2 and 4 weeks after the beginning of photostimulation and conforms with the approximately logarithmic growth described by Farner and Wilson (1957). It should be emphasized that photostimulation of Groups II through VII was begun on
,, 0
N2
NJ0 Competitor
ld
104
lo6
he1
FIG. 3. Competition of binding of r*%FSH to homogenates of the testis of the white-crowned sparrow by various gonadotropin preparations at different concentrations. Each reaction tube contained 131.OOOcpm (about 1.7 ng) of 9-FSH. 2.5 mg of the testicular tissue. and different amounts of the competitors. The volume of the reaction mixture was 0.14 ml. Incubation was performed at 37” for 3 hr. As the competitors, NIAMD rat FSH-I-3 (solid circles), NIAMD rat FSH-RP-1 (open circles), NIAMD rat LH-I-4 (solid squares), pregnant mare serum gonadotropin (solid triangles). and human chorionic gonadotropin (open triangles) were used.
FSH BINDING
BY SPARROW
TABLE 2 RELATIVE FSH POTENCIES OF VARIOUS GONADOTROP~N PREPARATIONS DETERMINED BY ‘THE RADIORECEPTORASSAY (CALCULATED FROM THE COMPETITION CURVES) AND BY BIOASSAY
0.4,.’
Gonadotropin preparation NIAMD-rat FSH-I-3 NIAMD-rat-FSH-RP-1 NIAMD-rat LH-RP-1 HCG NIAMD-rat LH-I-4
100 1 0.30 o.oooo4 not detectable
70 1 0.26 <0.02
:’ :’ . .--
al//f 0
a schedule (Table 1) that permitted the analyses of all groups within a period of 3 days and with the same lot of iodinated hormone which is stable for at least 1 week. The specific binding of FSH, expressed as a function of testicular weight, was high in the birds that had been kept on short days (8L 16D) and not photostimulated. It showed a slight increase after 1 week of the and then decreased photostimulation rapidly (Fig. 5). In contrast to specific binding, nonspecific binding, as a function of
; f ;
0 r s +l ..-*
I..
l.-- I
:
3 E 5I2-
Bioi3S&
a Expressed as the ratio to the potency of NIAMD-rat FSH-RF1 . b Calculated from the bioassay results given in the instructions of the NIAMDD Rat Pituitary Hormone Distribution Program.
I;
0.3-
FSH potency” Radioreceptor assay
447
TESTES
4.
./ .. ...e*’ 0 I 2 3 4 Wdu of photostlmubtion
s b -8 % 0” 5 * -6:: % % 40 0il%-20 ‘_ 5
0
FIG. 4. Effect of long-day treatment (20L 4D) on the weight of testes (solid circles and broken line) and the relative capacity (open circles and solid tine) of the specific binding of ?-rat FSH by the testes of the white-crowned sparrow. Mean values and their standard errors are indicated by circles and vertical bars, respectively.
testicular weight, showed only a slight and gradual decrease during the period of photostimulation. By plotting the specific binding per 2.5 mg of testicular tissue (Fig. 1, which shows the relation between binding and amount of tissue), the relative binding capacity of the entire testis was calculated. The capacity increased approximately in parallel with the increase of the
TABLE 3 MEAN SLOPE, ITS 95% CONFIDENCE LIMITS, AND INDEX OF PRECISION OF LINEAR PORTIONS OF THE COMPETITION CURVES OF NIAMD RAT FSH PREPARATIONS AND A HUMAN CHORIONIC GONADOTROPIN PREPARATION” Sample Rat FSH-I-3 Rat FSH-RP- 1 Rat LH-RP-1 HCG Weighted mean
Nb
Slope (b)
4 4 3 3
-4,794 -7,953 - 10,451 -4,972 -6,947
a Competition curves shown in Fig. 3. b Number of dose levels in the linear range of the curve.
Confidence limits -7,544 -11,748 -35,833 -7,611
and and and and
-2,044 -4,158 + 14,931 -2,333
-9,476 and -4,418
Index of precision 0.209 0.173 0.189 0.041 0.183
448
ISHII AND FARNER
:__-.*..: ....” , j 0
I” Weeks
2 3 of photostimulation
4
5
o
FIG. 5. Effect of long-day treatment (2OL 4D) on specific (open circles) and nonspecific (solid circles) binding of FSH by 2.5 mg of testicular tissue of the white-crowned sparrow. Mean bindings and their standard errors are indicated by circles and vertical bars, respectively. Testis weight is shown by the dotted line.
testicular weight for 3 weeks after the initiation of photostimulation (Fig. 4). However, the rate of increase became much slower or was almost nil after 3 weeks, while the testicular weight was still increasing at a high rate. DISCUSSION
The results of the competition experiments with various gonadotropin preparations show that the specific binding sites bound only FSH and not LH or contaminants in the gonadotropin preparations used. Competition for FSH binding induced by a large amount of HCG may be ascribable to the contamination of FSH in the HCG preparation used, since milligram quantities of HCG may contain nanogram quantities of FSH. The nonspecific binding shows a substantially lower tendency to saturate. The presence of two such types of binding has been described in the rat testis (Means and Vaitukaitis, 1972; Bhalla and Reichert, 1974; Miyachi and Inomata, 1974).
In the experiment with various incubation times, binding became maximal at about 3 hr. Bhalla and Reichert (1974) have also reported that maximum binding took place at 3 hr in the FSH binding with rat testis homogenate. On the other hand, Means and Vaitukaitis (1972) reported that only 1 hr was required for maximum binding. In the experiment on tissue concentration we found that the relation between binding and tissue concentration was nonlinear, which is consistent with the nonlinear relation found in the rat testis by Bhalla and Reichert (1974). In contrast, Miyachi and Inomata (1974) reported a linear relationship with slices of rat testis. However, their results showed that the relation would have become nonlinear had they used smaller amounts of tissue. The affinity (Kd = 0.78 nM) and capacity (7 fmoles/mg) of the specific binding sites of FSH in the testis of the white-crowned sparrow are similar to those reported for the rat (Kd = 0.68 and 7.0 ti, capacity being 62 and 3.5 fmoles/mg) by Bhalla and Reichert (1974) and by Means and Vaitukaitis (1972), respectively. Although temperature dependence and saturability remain to be demonstrated, the results of the competition experiment strongly suggest that the system used for FSH binding in the present study can be applied in a radioreceptor assay for avian FSH. It should be noted that temperature dependence and saturability have been demonstrated with a similar preparation of testis from the Japanese quail (Ishii, unpublished data). In the rat, Means and Vaitukaitis (1972) observed that the specific binding of FSH to the homogenate of a unit weight of the testis was greater in the immature (2800 cpm of 3H-labeled FSH/lOO mg of tissue) than in the mature rat (1700 cpm of 3Hlabeled FSH/lOO mg of tissue). Our results show a similar relation between resting and developing testis in relation to fully developed testis. However, the total capacity for binding by the entire testis was far
FSH BINDING
BY SPARROW TESTES
larger in the developed testis. Furthermore, the present study shows that the rapid increase of the binding capacity occurs in the earlier half of the period of testicular development induced by photostimulation. It is interesting to compare this finding with a recent finding in the Japanese quail by Follett (1976) that plasma FSH increases at an early phase of testicular development. The increase in FSH binding capacity with the development of the testis may be at least in part due to the increase in the area of membranes of the Sertoli cells by hypertrophy and/or hyperplasia. Recently, Ishii and Furuya (1975) and Ishii and Yamamoto (1976) have found that purified chicken FSH and ovine FSH specifically stimulate hypertrophy of the Sertoli cells of the chick testis. However, because of the very extensive changes in the cellular components of the testis during the course of photoperiodically induced growth and development, an explanation of these interesting changes in the number of concentration of binding sites must await correlative investigations using autoradiography. It would be interesting to identify the humoral (or other) factor that induces the increase in FSH binding capacity in the testis. Coincidence in the time of active secretion of FSH and the increase in FSH binding capacity in the testis, although these findings were obtained in different avian species, suggests a possibility that FSH itself induces FSH receptors. Recently, Posner et al. (1975) reported results suggesting that prolactin induces prolactin receptors in the liver of the rat. In contrast, Hepp et al. (1975) have suggested that insulin deficiency induces receptors in the liver of the Chinese hamster. Further study is required to reach definite conclusions concerning this problem. ACKNOWLEDGMENTS This study was supported in part by a grant from the Ministry of Education, Japan, and also by a grant from the Japanese Society for the Promotion of Sciences to Susumu Ishii and a grant (HD 06527) from the National Institutes of Health to Donald S. Farner.
449
We are grateful to Professor James McGinnis, Department of Animal Science, Washington State University, for supplying us with the Japanese quail used in developing some of the methods used in this investigation.
REFERENCES S., and J., Benveniste, R., Schwartz, Rabinowitz, D. (1974). Iodinated FSH: Components and their properties. Endocrinology 94, 952-958. Bhalla, V. K., and Reichert, L. E., Jr. (1974). Properties of follicle-stimulating hormone-receptor interactions. J. Biol. Chem. 249, 43-51. Catt, K. J., and Dufau, M. L. (1973). Interactions of LH and HCG with testicular gonadotropin receptors. In “Receptors for Reproductive Hormones, Advances in Experimental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 379-418. Plenum Press, New York/London. Farner, D. S., and Wilson, A. C. (1957). A quantitative examination of testicular growth in the white-crowned sparrow. Biol. Bull. 113,254-267. Follett, B. K. (1976). Plasma follicle-stimulating hormone during photoperiodically induced sexual maturation in male Japanese quail. J. Endocrinol. 69, in press. Follett, B. K., Farner, D. S., and Mattocks, P. W., Jr. (1975). Luteinizing hormone in the plasma of white-crowned sparrows (Zonotrichia leucophrys gambelii) during artificial photostimulation. Gen. Comp. Endocrinol. 26, 126-134. Greenwood, F. C., and Hunter, W. M. (1963). The preparation of ‘alI-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. Hepp, K. D., Langley, J., von Funcke, H. J., Renner, R., and Kemmler, W. (197.5). Increased insulin binding capacity of liver membranes from diabetic Chinese hamsters. Nature (London) 258, 154. Ishii, S., and Furuya, T. (1975). Effects of purified chicken gonadotropins on the chick testis. Gen. Comp. Endocrinol. 25, 1-8. Ishii, S., and Yamamoto, K. (1976). Demonstration of follicle stimulating hormone (FSH) activity in hypophyseal extracts of various vertebrates by the response of the Sertoli cells of the chick. Gen. Comp. Endocrinol. 29, 506-510. Lee, C. Y., and Ryan, R. J. (1973). Luteinizing hormone receptors in luteinized rat ovaries. In “Receptors for Reproductive Hormones, Advances in Experimental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 419-430. Plenum Press, New York/London. Means, A. R. (1973). Specific interaction of aH-FSH with rat testis binding sites. In “Receptors for Reproductive Hormones, Advances in ExperiBell,
450
ISHH AND FARNER
mental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 431-448. Plenum Press, New York/London. Means, A. R.. and Vaitukaitis, J. (1972). Peptide hormone “receptors”: Specific binding of aH-FSH to testis. Endocrinofogy 90, 39-46.
Miyachi, Y.. and Inomata, M. (1974). Specific binding of T-labeled human follicle stimulating hormone to testicular slice. Endocrinol. Jap. 21, 477-480. Posner, B. I., Kelly, P. A., and Friesen, H. G. (1975). Prolactin receptors in rat liver: Possible induction by prolactin. Science 188, 57-59.
AND
COMPARATIVE
Binding
ENDOCRINOLOGY
30, 443-450 (1976)
of Follicle-Stimulating Hormone by Homogenates of Testes of Photostimulated White-Crowned Sparrows, Zonotrichia leucophrys gambelii SUSUMU ISHII AND DONALD S. FARNER Department of Biology, School of Education, Waseda University, Tokyo 160, Japan, and Department of Zoology, University of Washington, Seattle, Washington 98195
Accepted July 16, 1976 Homogenates of testes of the white-crowned sparrow bind specifically with 1251-labeled rat FSH (follicle-stimulating hormone). The binding is competitively inhibited by unlabeled rat FSH and PMSG (pregnant mare serum gonadotropin) but not by purified rat LH (luteinizing hormone). Crude gonadotropin preparations also inhibit binding but the rates of inhibition are fully explained by amounts of FSH in these preparations. The testes of photosensitive birds with resting testes were found to bind about 4 x lo-i5 moles of ‘“I-labeled rat FSH per milligram of tissue. The binding per milligram of tissue decreases during photoperiodically induced testicular growth, although the total binding capacity increases during the first 3 weeks of photoperiodic stimulation. Thereafter it increases no further although the weight of the testes continues to increase rapidly. With further development, a specific and sensitive radioreceptor assay of avian FSH, using a testicular homogenate of the white-crowned sparrow as the receptor, may be possible.
It has been established in mammals that gonadotropins, as well as the other peptide hormones, exert their action by binding to specific receptors on the membrane of the target cells in gonads (e.g., Catt and Dufau, 1973; Lee and Ryan, 1973; Means, 1973). Accordingly, considerations of hormonal control of the gonad must take into account not only plasma levels of the hormones but also the binding of the hormones to the specific receptors in the target organ. The testes of photoperiodic birds in the photosensitive state undergo rapid growth under long-day conditions. In the white-crowned sparrow they attain a weight that is about 200 times the initial weight during 5 weeks of the photostimulation with 16 hr of light per day, 16L 8D (Follett et al., 1975). A remarkable increase in plasma gonadotropin during photostimulation has been reported in this species (Follett et al., 1975) and in the Japanese quail (e.g., Follett,
1976). However, to the best of our knowledge, no investigation has been published on the change in the gonadotropin binding by the avian gonad during photostimulation or even on the gonadotropin binding per se by the avian gonad. In this communication, we report that the testis of the white-crowned sparrow has specific binding sites for follicle stimulating hormone (FSH) and also that the binding capacity changes with the development of the testis under artificial photostimulation. MATERIALS
443 Copyright All rights
@ 1976 by Academic Press, inc. of reproduction in any form reserved.
AND METHODS
Adult male white-crowned sparrows were obtained with mist nets from migrating or wintering populations near Ellensburg, Kittitas County, and on the Sunnyside Game Refuge in Yakima County, both in central Washington, during the autumn of 1974 and late winter of 1975. After temporary confinement in large outdoor aviaries under natural conditions in Seattle, 38 were transferred to a constant-condition chamber (20 ? O.S”C, R.H. ca. 45%, 8-hr daily
444
ISHII
AND
photophase. 8L 16D) more than 6 months before they were used for the experiments described herein. They were divided at random into six groups: Group 1 (14 birds), Group II (8 birds), Groups III-VI (4 each): one bird was lost from Group III. Groups II-VI were exposed to 20-hr daily photophases (20L 4D) for 1, 2, 3, 4, and 5 weeks, respectively. At the end of the longday treatment, the birds were sacrificed for determination of the binding of FSH by the testes. Ten birds (Group VII), which had been captured in October 1975 and held in an outdoor aviary until 1 December, were transferred directly into a long-day chamber and were photostimulated for 8 days. All birds of Groups I-VII were sacrificed between 8 and 10 December, according to the schedule in Table 1. Binding experiments. After sacrifice by decapitation, testes of the birds were removed and weighed. All of the following procedures were performed under chilled conditions unless specified. Testes were homogenized (10 mg of tissue and 1 ml of medium) with a glass homogenizer with a Teflon rod by moving the rod up and down 10 times. The medium, Tris buffer-MgCl,BSA, consisted of 40 mM Tris-HCI buffer (pH 7.5) made 5 mM with MgCI, and 0.1% with bovine serum albumin. The testes of the birds in Groups I, II, and VII, respectively, were pooled for the homogenization. In Group III, testes of two or three birds were combined and homogenized. In Groups IV-VI, testes of each bird were homogenized separately. Homogenates were filtered through a layer of cheesecloth to remove fragments of tunica albuginea. Aliquots of the homogenates were then centrifuged at 5000 rpm in a refrigerated centrifuge for 10 min. The supernatant fluids were decanted and the sediments were resuspended with the buffer solution. The final concentration of the resuspended homogenate was adjusted to 2.5 mg of tissue/O.1 ml in ah experiments except the one that
FARNER
required a series of concentrations of tissue. Then 0.1 ml of homogenate, 20 ~1 of a radioiodinated rat follicle-stimulating hormone (i251-FSH) solution and 20 ~1 of a cold rat FSH solution, or the vehicle alone, were mixed in a glass tube coated with silicone and incubated at 37” with continuous shaking. The incubation time was 5 hr in all of the experiments except the one in which incubation time was varied. The radioiodinated hormone and cold hormone were dissolved in 10 mM phosphate buffer made up to 0.15 M with NaCl and with 0.1% BSA-20 pl of ‘Z51-FSH (13-14 x 104 cpm) and 20 ~1 of cold FSH solution containing 1.2 pg of the cold FSH. In competition experiments different amounts of various gonadotropin preparations were used instead of the cold FSH. At the end of the incubation 1 ml of chilled Tris buffer-MgCl,-BSA was added to each reaction tube, and the reaction tubes were centrifuged at 5000 rpm for 10 min. The sediments thus obtained were washed twice by resuspension and centrifugation. The sediments obtained after the final centrifugation were counted for radioactivity of the lz51-FSH bound to sedimented particles in an automatic gamma counter (Nuclear Chicago). The radioactivity of the sediment of the tube that contained no cold hormone was expressed as the total binding and that of the tube containing 1.2 pg of cold FSH as the nonspecific binding. The difference between the total and nonspecific binding was defined as the specific binding. Because of the limited amount of purified hormones available and the rather low coefficient of variation in the determination of the binding (less than 0.12 in a preliminary experiment), only single determinations were used throughout the experiments. Hormone preparations. NIAMD rat FSH-I-3 was used for radioiodination. Hormone preparations used for the competition experiments were NIAMD rat
TABLE
1
PHOTOSTIMULATION SCHEDULE AND MEAN BODY WEIGHT AT SACRIFICE WHITE-CROWNED SPARROWS IN EACH EXPERIMENTAL GROUP
Group I II III IV V VI VII
Number of birds 14 8 3 4 4 4 10
Photostimulation
(2OL 4D)
Period
Days
I- 8 Dec. 24 Nov.- 8 Dec. 19 Nov.-l0 Dec. 12 Nov.-l0 Dec. 5 Nov.-l0 Dec. I- 9 Dec.
0 7 14 21 28 35 8
a The increase in terminal body weight is the result of photoperiodically of this species.
OF
Terminal mean body weight -c SEM (g)” 26.1 27.1 29.1 29.5 30.0 27.5 28.6
i- 0.7 + 0.7 T’- 1.3 ‘- 1.0 2 2.1 +- 0.7 2 0.8
induced fat deposition characteristic
FSH BINDING
BY
LH-RP-1, NIAMD rat LH-I-4, pregnant mare serum gonadotropin (PMSG) (Sigma), and human chorionic gonadotropin (HCG) (Parke Davis). HCG was first dialyzed against phosphate buffer-saline overnight in order to remove the benzethonium chloride present as a preservative. Iodination procedure. The iodination of purified rat FSH was performed using lactoperoxidase as the catalytic agent. To the reaction tube containing 2.5 Mg of FSH dissolved in 25 ~1 of phosphate buffer-saline were added 30 bl of 0.4 M acetate buffer (pH 5.6), 200 mg of lactoperoxidase (Sigma) dissolved in 5 ~1 of 0.1 M acetate buffer (pH 5.6), 0.5 mCi of carrier-free NaLzSI (Radiochemical Centre, Amersham) dissolved in 5 ~1 of 0.1 N NaOH, and 5 ~1 of a solution containing 75 ng of HZ02. The mixture was held at room temperature for 15 min during which 5 ~1 of the HZOZ solution was added at 5-min intervals (three times in total). The reaction mixture was then transferred to a column of Sephadex G-75. Preparation of the column and separation of ‘*%FSH from inorganic iodide by the column were performed according to the instructions for the radioimmunoassay of rat follicle stimulating hormone, Rat Pituitary Hormone Distribution Program, NIAMDD. The recovery of the hormone in the eluate from the column was estimated according to Greenwood and Hunter (1963). The specific activity of the labeled FSH was estimated to be 45 mCi/mg. Offhand, this activity appears somewhat high, raising the possibility of damage of the hormone. However, comparisons of low- and highactivity preparations on testes from Japanese quail (Ishii, unpublished) revealed no significant differences in results. Furthermore, Bell et al. (1974) have used, with satisfactory results, radioiodinated FSH with a specific activity as high as 60 mCi/mg.
SPARROW
445
TESTES
0
2.5 Tissue
5.0
weight
(me)
FIG. 1. Binding of ‘%FSH to homogenates of the testis of the white-crowned sparrow at varying concentrations of tissue. Each reaction tube contained 13.1 x 104 cpm of *?-FSH; 1.25, 2.5, or 5.0 mg of the testicular tissue; and 1.2 pg of cold FSH or vehicle alone. Incubation was performed at 37” for 3 hr. See text for definitions of total (broken line and solid circles) and specific (continuous line and open circles) bindings.
Total
RESULTS
Binding of lz5Z-FSH as function of incubation time and tissue concentration. Homogenates containing 2.5 mg of testicular tissue from Group I and lz51-FSH were incubated with or without an excess amount of cold FSH for 1, 2, and 3 hr, and the total and nonspecific bindings were measured (Figs. 1 and 2). Both types of binding proceeded rapidly for about 1 hr and then increased gradually for the next 2 hr (Fig. 2), reaching an equilibrium in approximately 3 hr. The ratio of specific to total binding at equilibrium was 53%. Homogenates containing 2.5, 5.0, and 10.0 mg of tissue from Group I were incubated with 1251-FSH and an excess amount
Incubation
time
(hr)
2. Binding of ‘*%I-FSH to homogenates of the testis of the white-crowned sparrow at different times of incubation. Each reaction tube contained 13.1 x 104 cpm of Y-FSH, 2.5 mg of the tissue, and 1.2 /Ig of cold FSH or vehicle alone. Incubation was performed at 37” for 1, 2, or 3 hr. See text for definitions of total (broken line and solid circles) and specific (continuous line and open circles) bindings. FIG.
446
ISHII AND FARNER
of cold FSH or with ‘“51-FSH alone for 3 hr, and the total and nonspecific bindings were measured. Both total and specific bindings were nonlinear as a function of the concentration of the homogenate under the conditions used in this experiment (Fig. 1). The ratio of specific to total binding at equilibrium was 53%. Competition of binding of ‘251-FSH by various gonadotropin preparations. Homogenates of testicular tissue derived from Group VII were incubated with 1251FSH with increasing amounts of the various gonadotropin preparations. NIAMD rat FSH-I-3, the preparation used for the labeled hormone, competed with the binding of the labeled hormone in small amounts (Fig. 3); 50% competition was obtained at about 3 ng. NIAMD rat FSH-RP1, NIAMD rat LH-RP-1, and PMSG also showed clear competition at moderate amounts (Fig. 3). The slopes of the competition curves were similar among these hormone preparations. NIAMD rat LH-I-4 showed essentially no competition even when present in large amounts, ca. 2 pg. HCG competed only at extremely large amounts, ca. 1 mg (Fig. 3). The relative FSH potencies of the NIAMD gonadotropin preparations and
d
HCG were calculated from distances on the competition curves at about the 50% competition level and are given in Table 2. The statistical analyses of the competition curves in Fig. 3 are given in Table 3. The curves can be regarded as parallel. The relative FSH potencies thus obtained coincide with FSH potencies determined by bioassay, as shown in the instructions from NIAMDD. A Scatchard plot of the data from the competition experiment with NIAMD-rat FSH-I-3 was performed after removing the two marginal values. The dissociation constant at equilibrium and the capacity for binding obtained by the Scatchard plot were 0.78 nM and 7.0 fmoles of FSH/mg of tissue, respectively. Change in the binding with development of testes. The weight of the testes of the photostimulated birds continued to increase through the stimulation period and became maximal at the fifth week (Fig. 4). The increase was especially marked between 2 and 4 weeks after the beginning of photostimulation and conforms with the approximately logarithmic growth described by Farner and Wilson (1957). It should be emphasized that photostimulation of Groups II through VII was begun on
,, 0
N2
NJ0 Competitor
ld
104
lo6
he1
FIG. 3. Competition of binding of r*%FSH to homogenates of the testis of the white-crowned sparrow by various gonadotropin preparations at different concentrations. Each reaction tube contained 131.OOOcpm (about 1.7 ng) of 9-FSH. 2.5 mg of the testicular tissue. and different amounts of the competitors. The volume of the reaction mixture was 0.14 ml. Incubation was performed at 37” for 3 hr. As the competitors, NIAMD rat FSH-I-3 (solid circles), NIAMD rat FSH-RP-1 (open circles), NIAMD rat LH-I-4 (solid squares), pregnant mare serum gonadotropin (solid triangles). and human chorionic gonadotropin (open triangles) were used.
FSH BINDING
BY SPARROW
TABLE 2 RELATIVE FSH POTENCIES OF VARIOUS GONADOTROP~N PREPARATIONS DETERMINED BY ‘THE RADIORECEPTORASSAY (CALCULATED FROM THE COMPETITION CURVES) AND BY BIOASSAY
0.4,.’
Gonadotropin preparation NIAMD-rat FSH-I-3 NIAMD-rat-FSH-RP-1 NIAMD-rat LH-RP-1 HCG NIAMD-rat LH-I-4
100 1 0.30 o.oooo4 not detectable
70 1 0.26 <0.02
:’ :’ . .--
al//f 0
a schedule (Table 1) that permitted the analyses of all groups within a period of 3 days and with the same lot of iodinated hormone which is stable for at least 1 week. The specific binding of FSH, expressed as a function of testicular weight, was high in the birds that had been kept on short days (8L 16D) and not photostimulated. It showed a slight increase after 1 week of the and then decreased photostimulation rapidly (Fig. 5). In contrast to specific binding, nonspecific binding, as a function of
; f ;
0 r s +l ..-*
I..
l.-- I
:
3 E 5I2-
Bioi3S&
a Expressed as the ratio to the potency of NIAMD-rat FSH-RF1 . b Calculated from the bioassay results given in the instructions of the NIAMDD Rat Pituitary Hormone Distribution Program.
I;
0.3-
FSH potency” Radioreceptor assay
447
TESTES
4.
./ .. ...e*’ 0 I 2 3 4 Wdu of photostlmubtion
s b -8 % 0” 5 * -6:: % % 40 0il%-20 ‘_ 5
0
FIG. 4. Effect of long-day treatment (20L 4D) on the weight of testes (solid circles and broken line) and the relative capacity (open circles and solid tine) of the specific binding of ?-rat FSH by the testes of the white-crowned sparrow. Mean values and their standard errors are indicated by circles and vertical bars, respectively.
testicular weight, showed only a slight and gradual decrease during the period of photostimulation. By plotting the specific binding per 2.5 mg of testicular tissue (Fig. 1, which shows the relation between binding and amount of tissue), the relative binding capacity of the entire testis was calculated. The capacity increased approximately in parallel with the increase of the
TABLE 3 MEAN SLOPE, ITS 95% CONFIDENCE LIMITS, AND INDEX OF PRECISION OF LINEAR PORTIONS OF THE COMPETITION CURVES OF NIAMD RAT FSH PREPARATIONS AND A HUMAN CHORIONIC GONADOTROPIN PREPARATION” Sample Rat FSH-I-3 Rat FSH-RP- 1 Rat LH-RP-1 HCG Weighted mean
Nb
Slope (b)
4 4 3 3
-4,794 -7,953 - 10,451 -4,972 -6,947
a Competition curves shown in Fig. 3. b Number of dose levels in the linear range of the curve.
Confidence limits -7,544 -11,748 -35,833 -7,611
and and and and
-2,044 -4,158 + 14,931 -2,333
-9,476 and -4,418
Index of precision 0.209 0.173 0.189 0.041 0.183
448
ISHII AND FARNER
:__-.*..: ....” , j 0
I” Weeks
2 3 of photostimulation
4
5
o
FIG. 5. Effect of long-day treatment (2OL 4D) on specific (open circles) and nonspecific (solid circles) binding of FSH by 2.5 mg of testicular tissue of the white-crowned sparrow. Mean bindings and their standard errors are indicated by circles and vertical bars, respectively. Testis weight is shown by the dotted line.
testicular weight for 3 weeks after the initiation of photostimulation (Fig. 4). However, the rate of increase became much slower or was almost nil after 3 weeks, while the testicular weight was still increasing at a high rate. DISCUSSION
The results of the competition experiments with various gonadotropin preparations show that the specific binding sites bound only FSH and not LH or contaminants in the gonadotropin preparations used. Competition for FSH binding induced by a large amount of HCG may be ascribable to the contamination of FSH in the HCG preparation used, since milligram quantities of HCG may contain nanogram quantities of FSH. The nonspecific binding shows a substantially lower tendency to saturate. The presence of two such types of binding has been described in the rat testis (Means and Vaitukaitis, 1972; Bhalla and Reichert, 1974; Miyachi and Inomata, 1974).
In the experiment with various incubation times, binding became maximal at about 3 hr. Bhalla and Reichert (1974) have also reported that maximum binding took place at 3 hr in the FSH binding with rat testis homogenate. On the other hand, Means and Vaitukaitis (1972) reported that only 1 hr was required for maximum binding. In the experiment on tissue concentration we found that the relation between binding and tissue concentration was nonlinear, which is consistent with the nonlinear relation found in the rat testis by Bhalla and Reichert (1974). In contrast, Miyachi and Inomata (1974) reported a linear relationship with slices of rat testis. However, their results showed that the relation would have become nonlinear had they used smaller amounts of tissue. The affinity (Kd = 0.78 nM) and capacity (7 fmoles/mg) of the specific binding sites of FSH in the testis of the white-crowned sparrow are similar to those reported for the rat (Kd = 0.68 and 7.0 ti, capacity being 62 and 3.5 fmoles/mg) by Bhalla and Reichert (1974) and by Means and Vaitukaitis (1972), respectively. Although temperature dependence and saturability remain to be demonstrated, the results of the competition experiment strongly suggest that the system used for FSH binding in the present study can be applied in a radioreceptor assay for avian FSH. It should be noted that temperature dependence and saturability have been demonstrated with a similar preparation of testis from the Japanese quail (Ishii, unpublished data). In the rat, Means and Vaitukaitis (1972) observed that the specific binding of FSH to the homogenate of a unit weight of the testis was greater in the immature (2800 cpm of 3H-labeled FSH/lOO mg of tissue) than in the mature rat (1700 cpm of 3Hlabeled FSH/lOO mg of tissue). Our results show a similar relation between resting and developing testis in relation to fully developed testis. However, the total capacity for binding by the entire testis was far
FSH BINDING
BY SPARROW TESTES
larger in the developed testis. Furthermore, the present study shows that the rapid increase of the binding capacity occurs in the earlier half of the period of testicular development induced by photostimulation. It is interesting to compare this finding with a recent finding in the Japanese quail by Follett (1976) that plasma FSH increases at an early phase of testicular development. The increase in FSH binding capacity with the development of the testis may be at least in part due to the increase in the area of membranes of the Sertoli cells by hypertrophy and/or hyperplasia. Recently, Ishii and Furuya (1975) and Ishii and Yamamoto (1976) have found that purified chicken FSH and ovine FSH specifically stimulate hypertrophy of the Sertoli cells of the chick testis. However, because of the very extensive changes in the cellular components of the testis during the course of photoperiodically induced growth and development, an explanation of these interesting changes in the number of concentration of binding sites must await correlative investigations using autoradiography. It would be interesting to identify the humoral (or other) factor that induces the increase in FSH binding capacity in the testis. Coincidence in the time of active secretion of FSH and the increase in FSH binding capacity in the testis, although these findings were obtained in different avian species, suggests a possibility that FSH itself induces FSH receptors. Recently, Posner et al. (1975) reported results suggesting that prolactin induces prolactin receptors in the liver of the rat. In contrast, Hepp et al. (1975) have suggested that insulin deficiency induces receptors in the liver of the Chinese hamster. Further study is required to reach definite conclusions concerning this problem. ACKNOWLEDGMENTS This study was supported in part by a grant from the Ministry of Education, Japan, and also by a grant from the Japanese Society for the Promotion of Sciences to Susumu Ishii and a grant (HD 06527) from the National Institutes of Health to Donald S. Farner.
449
We are grateful to Professor James McGinnis, Department of Animal Science, Washington State University, for supplying us with the Japanese quail used in developing some of the methods used in this investigation.
REFERENCES S., and J., Benveniste, R., Schwartz, Rabinowitz, D. (1974). Iodinated FSH: Components and their properties. Endocrinology 94, 952-958. Bhalla, V. K., and Reichert, L. E., Jr. (1974). Properties of follicle-stimulating hormone-receptor interactions. J. Biol. Chem. 249, 43-51. Catt, K. J., and Dufau, M. L. (1973). Interactions of LH and HCG with testicular gonadotropin receptors. In “Receptors for Reproductive Hormones, Advances in Experimental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 379-418. Plenum Press, New York/London. Farner, D. S., and Wilson, A. C. (1957). A quantitative examination of testicular growth in the white-crowned sparrow. Biol. Bull. 113,254-267. Follett, B. K. (1976). Plasma follicle-stimulating hormone during photoperiodically induced sexual maturation in male Japanese quail. J. Endocrinol. 69, in press. Follett, B. K., Farner, D. S., and Mattocks, P. W., Jr. (1975). Luteinizing hormone in the plasma of white-crowned sparrows (Zonotrichia leucophrys gambelii) during artificial photostimulation. Gen. Comp. Endocrinol. 26, 126-134. Greenwood, F. C., and Hunter, W. M. (1963). The preparation of ‘alI-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. Hepp, K. D., Langley, J., von Funcke, H. J., Renner, R., and Kemmler, W. (197.5). Increased insulin binding capacity of liver membranes from diabetic Chinese hamsters. Nature (London) 258, 154. Ishii, S., and Furuya, T. (1975). Effects of purified chicken gonadotropins on the chick testis. Gen. Comp. Endocrinol. 25, 1-8. Ishii, S., and Yamamoto, K. (1976). Demonstration of follicle stimulating hormone (FSH) activity in hypophyseal extracts of various vertebrates by the response of the Sertoli cells of the chick. Gen. Comp. Endocrinol. 29, 506-510. Lee, C. Y., and Ryan, R. J. (1973). Luteinizing hormone receptors in luteinized rat ovaries. In “Receptors for Reproductive Hormones, Advances in Experimental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 419-430. Plenum Press, New York/London. Means, A. R. (1973). Specific interaction of aH-FSH with rat testis binding sites. In “Receptors for Reproductive Hormones, Advances in ExperiBell,
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mental Medicine and Biology” (B. W. O’Malley, and A. R. Means, eds.), Vol. 36, pp. 431-448. Plenum Press, New York/London. Means, A. R.. and Vaitukaitis, J. (1972). Peptide hormone “receptors”: Specific binding of aH-FSH to testis. Endocrinofogy 90, 39-46.
Miyachi, Y.. and Inomata, M. (1974). Specific binding of T-labeled human follicle stimulating hormone to testicular slice. Endocrinol. Jap. 21, 477-480. Posner, B. I., Kelly, P. A., and Friesen, H. G. (1975). Prolactin receptors in rat liver: Possible induction by prolactin. Science 188, 57-59.