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Estradiol binding activity in epididymal cytosol of the turtle, Chrysemys picta
GENERAL
AND
COMPARATIVE
Estradiol
ENDOCRINOLOGY
51,
Binding Activity
61-65 (1983)
in Epididymal
Chrysemys JEAN-PIERRE Department
DUFAURE,~
PAUL
Cytosol of the Turtle,
picta MAK, AND IAN P. CALLARD
of Biology, Boston University, Boston, Massachusetts 02215 Accepted August 5, 1982
Estrogen binding activity was investigated in the epididymis of the turtle, Chrysemys picta using DNA-cellulose affinity chromatography. A component binding estradiol-17P specifically with high affinity (&:8.0 x lo-lo M) and limited capacity (20 fmol/mg protein) was demonstrated in the epididymal cytosol. In addition, binding of estradiol-17P was sensitive to excess (loo-fold) diethylstilbestrol or natural estrogens (estradiol-17& estrone, and estriol) but not to progesterone or androgens (testosterone and Sa-dihydrotestosterone). The specific estrogen binding macromolecules eluted from DNA-cellulose columns sedimented at 4-5 S in linear 5-20% sucrose gradients. These characteristics suggest the presence of an estrogen receptor in this androgen target organ.
(Mak et al., 1982) of turtles from bath Androgen target tissues of the genital tract are influenced by estrogens in the dog sexes, and in the testis (Mak et al., submit(Huggins and Clark, 1940; Tunn et al., ted). The epididymis of the turtle shows 1979), rat (Feycl-Cabanes et al., 1977; well defined seasonal changes in weight and Thompson et al., 1979), calf (Kroes and histology (Callard, I. P., unpublished obTeppema, 1972), and guinea pig (Belis et servations) and in the lizard, Lacerta vivial., 1977). Specifically, estrogens can pro- para, binding of [H3]estradiol to epidihynial mote growth of the prostate and act prefer- cell nuclei has been demonstrated by autoentially on the fibromuscular stroma radiography (Dufaure et al., 1978). Th? purpose of this study was to advance our un(Feycl-Cabanes et al., 1977; Thompson et derstanding of the possible role of estrogen al., 1979). Consistent with these actions, cytoplasmic receptor proteins for estradiol in regulating seasonal breeding by identifyhave been described in prostate glands of ing a putative estrogen receptor in turtle the dog (Chairsiri et al., 1978; Robinette et epididymal cytosol. al., 1978; Dube et al., 1979), the rat (JungMATERIALS AND METHODS blut et al., 1971; Armstrong and Bashirelahi, 1974, 1978; Dubois et al., 1980; GinsSexually mature male Chrysemys picta were obburg et al., 1980), and some other species. tained from Nasco-Steinhilber, Inc. (For+ Atkinson, Wis.) from October through December and were mainAmong the Reptilia, estrogen receptor tained in the laboratory as previously described (Calproteins have been identified in estrogen lard et al., 1978). target tissues (oviduct) of the lizard (Botte Animals were killed by decapitation and all subseet al., 1974) and turtle (Salhanick et al., quent procedures were performed at 0-4°C. Tissues 1979). In addition, putative estrogen recep- were carefully washed in extraction buffer, minced, and homogenized in 4 vol (wtivol) buffer tors have been demonstrated in the liver weighed, with a glass-glass Potter Elvehjem homogenizer, $he (Heisermann et al., 1980) and the brain homogenate was ceritrifuged at 1400s for 20 Fin and cytdsol was prepared by centrifqation.of the r@lting supernate at 100,OOOgfor 1 hr. The protein concentration of cytosol was determined by ‘the method of Lowry et al., (19.51) as modified by Geiger and Beasman (1972) with BSA as the standard.
1 Present ‘address: Lab. Biologie Cellulaire et G&Ctique, Complexe Scientifique des Cezeaux, Universitb de Clermont-Fd 11, B.P. 45 63170 AubSre, France. 61
0016-6480183 $1.50 Copyright AU rights
@ 1983 by Academic Press, Inc. of reproduction in any form reserved.
62
DUFAURE,
L
I
2
I
4
6
I
1
6
No
MAK,
I
AND CALLARD (2-3 mg protein) were chromatographed onto columns by gravity and allowed to remain in contact with the DNA cellulose for 30 ruin at 4°C. The columns were then washed with 6-7 vol of buffer to remove any components not adhering to DNA cellulose and the labeled steroid dissolved in 50 m&’ NaCl buffer was subsequently loaded to the columns by gravity. For incubation, columns were sealed and kept at 23°C for 45 min. After returning to a 4°C cold room for 15 min, unbound steroid was removed by washing the columns with lo-12 vol of buffer containing 50 mM NaCl. DNA adhering components labeled with [3H]estradiol were eluted stepwise with buffer containing 400 mM NaCl and 1 mg BSA per ml. 0.5 ml fractions were collected and counted using a scintillation cocktail (750 ml Triton X-114, 2250 ml xylene, 9.0 g PPO, 0.75 g POPOP). Sucrose gradient centrifugation. Estrogen binding components eluted from DNA-cellulose columns were mixed with 5 ~1 [methyl-‘T] BSA and layered on 10 ml, 5-20% linear sucrose gradient prepared in TEGM buffer with 400 m&4 NaCl. Gradients were centrifuged at 240,OOOg for 20 hr (IEC centrifuge B-60, rotor SW283, at 4°C). Fractions (16 drops each) were collected from the bottom of the tube directly into counting
10 12 FRACTION
1. DNA-cellulose affinity chromatography of turtle epididymal cytosol. Samples (2.16 mg protein) were chromatographed onto 2 ml DNA-cellulose columns and incubated at 23°C for 45 mitt with 5 nit4 [3H]estradiol f loo-fold excess nonradioactive diethylstilbestrol (DES) as described under Methods. Bound radioactivity which adhered to the columns was eluted stepwise with TEMG buffer containing 400 n&f NaCl and 1.0 mg BSA per ml. Radioactivity was measured in 0.5 ml fractions after addition of 4.0 ml scintillation cocktail. 0, 5 nM [3H]estradiol; & 5 nM [3H]estradiol plus loo-fold excess nonradioactive DES. FIG.
Chemicals. Extraction buffer contained 10 n&4 Tris-HCl (pH 7.8), 1 mM Na, EDTA, 1 mM p-mercaptoethanol, 10% (vol/vol) glycerol (TEGM) and 50 mM NaCl. The molarity of NaCl was varied during chromatography from 50 to 400 mll/l; BSA (Pentex bovine albumin) from Miles Laboratory, Inc.: [2,4,6,73H]estradiol-17P (90- 110 Ci/mmol) and [r4C]BSA from New England Nuclear Corporation. Stock solutions of steroids were prepared in absolute ethanol and kept at -20°C. DNA-cellulose
afJinity chromatography. DNA-cellulose was prepared according to the method of Alberts and Herrick (1970) as modified by Fox and Pardee (1971). Briefly, 2 ml columns of DNA cellulose were equilibrated with 10 vol of buffer containing 50 mM NaCl and BSA (0.2 mgiml). Samples of cytosol
G +
FIG. 2. Competition analysis for estrogen binding sites. Equal volumes of cytosol(l.62 mg protein) were applied to DNA-cellulose columns and incubated with 3 nM [3H]estradiol -C lOO-fold excess different nonradioactive steroids. Other conditions were as in Fig. 1. The values of the different peaks of radioactivity corresponding to the binding complexes were calculated from the elution pattern and by subtracting the background. E, , estradiol-17p; E,, estrone; E,, estriol; DES, diethylstilbestrol; Pg, progesterone; T, testosterone; DHT, 5a dihydrotestosterone.
ESTRADIOL
BINDING
IN
TURTLE
63
EPIDIDYMIS
$:8x
IO-10 M
FIG. 3. Saturation analysis of estrogen binding in turtle epididymal cytosol using DNA-cellulose affinity chromatography. Samples of cytosol(2 mg protein) were chrumatographed onto DNA-celhtlose columns as described under Methods. Columns were incubated with [3H]estradiol from 0.5-5.0 r&f to determine total binding (A). Additional columns were incubated with 1.0,3.2, and 5.0niVQ3H]estradiol in the presence of IOO-fold excess nonradioactive dietbylstilbestrol (DES) to determine nanspecific binding (A). Specific binding (e) was obtained by subtracting nonspecific from total. Scatchard plot analysis of binding data revealed Kd: 8.0 x 10-W and m:.ximal binding sites: 20.6 fmok’mg protein.
vials. [‘*C]BSA (NEN) served as internal marker for each gradient.
RESULTS AND DISCUSSION
The data suggest the presence of a putative estrogen receptor in the cytosol of turtle epididymis. The elution pattern of DNAcellulose affinity chromatography for the estrogen binding component is illustrated in BSA
Top
5
IO NO
15
20
“&I
FRACTION
FIG. 4. Sedimentation analysis of estrogen binding components eluted from DNA cellulose. 0.8 ml of the 0.4 M NaCl DNA eluate was mixed with 5 ~1 l*C (Bovine Serum Albumin) and layered on a 10 ml, S-20% linear sucrose gradient as described under Methods. The arrow indicates the position of the internal marker [‘T]BSA (4.6 S).
Fig. 1. Such binding is specific, being displaced with a loo-fold excess of unlabeled competitor (diethylstilbestrol). The hormonal specificity was demonstrated by incubating the columns with r3HJestradiol (3 nM) plus lOO-fold excess of various unlabeled steroid competitors. As shown in Fig. 2, [3H]estradiol binding was efficiently displaced by unlabeled estradiol and diethylstilbestrol (DES) but not by testosterone, Sa-dihydrotesterone, or progesterone. Estrone and estriol were less effective than estradiol or DES. Tissue specificity was demonstrated in previous experiments which showed that bound radioactivity could not be detected in plasma, kidney, muscle, and lung (see Mak et al., 1982). Scatchard plot analysis of the binding data obtained after incubation with [3H&stradiol between 0.4 to 5.0 n&I L IOO-fold excess ‘DES gave values confirming high affinity (Kd: 8& x lo+’ M) and low capacity (20.6 fmolimg protein, Fig. 3). The estrogen binding macromolecules eluted from DNA celhuose had a sedimentation coefficient of 4,5 S
64
DUFAURE,
MAK,
(Fig. 4). Similar macromolecular components have also been demonstrated in the testicular cytosol of turtle (Mak et al., submitted). According to its binding affinity, specificity, and physicochemical properties, the estrogen binding activity of the turtle epididymis appears related to a receptor species rather than to a sex-steroid-binding protein originating from the plasma (Salhanick and Callard, 1980). Previous studies have demonstrated the androgen dependency of lizard epididymal growth and secretion (Gigon-Depeiges and Dufaure, 1977; Depeiges et al., 1982) and binding of labeled estradiol-17P to epididymal cell nuclei has been demonstrated in the same species (Lacerta vivipara) (Dufaure and Chambon, 1978). The exact function of estrogen receptors in the turtle epididymis is not known. However, preliminary data showed that receptor levels fluctuated during the annual reproductive cycle. Since turtle epididymis and plasma testosterone show a well defined seasonal cycle (Callard, I. P., unpublished observations), it. is possible that both estrogens and androgens play an important role in sperm maintenance or maturation in such seasonally breeding species. REFERENCES Alberts, B., and Herrick, G. (1970). DNA-cellulose chromatography. Meth. Enzymol. 21, 198-271. Armstrong, E. G., and Bashirelahi, N. (1974). A specific binding protein for 17P-estradiol in retired breeder rat ventral prostate. Biochem. Biophys. Res. Commun. 61, 628-634. Armstrong, E. G., and Bashirelahi, N. (1978). Determination of the binding properties of estradiol-17p within the cytoplasmic and nuclear fractions of rat ventral prostate. J. Steroid Biochem. 9, 507-513. Belis, J. A., Blume, C. D., and Mawhinney, M. G. (1977). Androgen and estrogen binding in male guinea-pig accessory sex organs. Endocrinology 101, 726-740. Botte, V., Granata, G., and Cristofaro, C. (1974). Interaction of 17P-estradiol and testosterone with the oviduct of the lizard Lacerta sicula. J. Steroid Biochem. 5, 687-691. Callard, I. P., Lance, V., Salhanick, A. R., and Barad, D. (1978). The annual ovarian cycle of Chrysemys
AND CALLARD Correlated changes in plasma steroids and parameters of vitellogenesis. Gen. Comp. Endo-
picta:
crinol.
35, 245-2.57.
Chaisiri, N., Valotaire, Y., Bronwen, A., Evans, J., and Pierrepoint, C. G. (1978). Demonstration of cytoplasmic receptor protein for oestrogen in the canine prostate gland. J. Endocrinol. 78, 131139. Depeiges, A., Betail, G., and Dufaure, J. P. (1982). Time course of appearance in viva and in vitro of a specific epididymal protein controlled by testosterone. Bio. Cell. 42, (in press). Dube, J. Y., Lesage, R., and Tremblay, R. R. (1979). Estradiol and progesterone receptors in dog prostate cytosol. J. Steroid Biochem. 10, 459-466. Dubois, R., Dube, .I. Y., and Tremblay, R. R. (1980). Presence of three different estradiol binding proteins in rat prostate cytosol. J. Steroid Biochem. 13, 1467-1471. Dufaure, J. P., and Chambon, M. (1978). Etude histoautoradiographique dun organecible de la testosterone, l’epididyme de lezard (Lacerta viviparu) apres administration de 17@-oestradiol 3H. C. R. Sot. Biol. 172, 1127-1130. Feycl-Cabanes, T., Robel, P., and Baulieu, E. E. (1977). Effets conjoints de la testosterone et de l’oestradiol sur le lobe ventral de la prostate du rat en cultures organotypique. C. R. Acad. Sci. (Paris) SPr. D 285, 1119-1122. Fox, T. O., and Pardee, A. B. (1971). Proteins made in the mammalian cell cycle. J. Biol. Chem. 246, 6159-6165. Geiger, P. J., and Bessman, S. P. (1972). Protein determination by Lowry’s method in the presence of sulthydryl reagents. Anal. Biochem. 49,467-473. Gigon-Depeiges, A., and Dufaure, J. P. (1977). Secretory activity of the lizard epididymis and its control by testosterone. Gen. Comp. Endocrinol. 33, 473-479. Ginsburg, M., Jung-Testas, I., and Baulieu, E. E. (1980). Specific high-affinity oestradiol binding in rat ventral prostate. J. Endocrinol. 87, 285-292. Heisermann, G. J., Ho, S. M., and Callard, I. P. (1980). Estrogen binding activity in the liver of the Eastern painted turtle, Chrysemys picta. Amer. Zool. 20, Abstr. 360. Huggins, C., and Clark, P. J. (1940). Quantitative studies on prostatic secretions. II. The effect of castration and of estrogen injeetion on the normal and on the hyperptastic prostate glands of dogs. J. Exp.
Med.
72,747-761.
Jungblut, P. W., Hughes, S. F., Gorlich, L., Gowers, V., and Wagner, R. K. (1971). Simultaneous occurrence of individual estrogen and androgen receptors in female and male target organs. HoppeSeyler’s Z. Physiol. Chem. 352, 1603-1610. Kroes, R., and Teppema, J. S. (1972). Development
ESTRADIOL
BINDING
and restitution of squamous metaplasia in the calf prostate after a single estrogen treatment. Exp. Mol. Pafhol. 16, 286-301. Lowry, 0. H., Roseborough, N. J., Fat-r, A. L., and Randall, R. J. (1951). Protein measurements with folinphenol reagent. J. Biol. Chem. 193, 265-279. Mak, P., Ho, S. M., and Callard, I. P. (1982). Estrogen receptors in the turtle brain. Brain Res. 231, 63Mak, P., Ho, S. M., and Callard, I. P. Characterization of an estrogen receptor species in the turtle testis. Gen. Camp. Endocrinol. (in press). Robinette, C. L., Blume, C. D., and Mawhinney, M. G. (1978). Andropbilic and estrophilic molecules in canine prostate glands. Invest. Ural. 15, 425-431.
Salhanick, A. R., Vito, C. C., Fox, T. O., and Callard,
IN
TURTLE
EPIDIDYMIS
65
I. P. (1979). Estrogen binding proteins in the oviduct of the turtle, Chrysemys picta: Evidence for a receptor species. Endocrinology 105, DSS1395. Salhanick, A. R., and Callard, I. P. (1980). A sexsteroid-binding protein in the plasma of the fresh water turtle, Chrysemys picta. Gen. Comp. Endocrinoi. 42, 163-166. Thompson, S. A., Rowley, D. R., and Heidger, P. M. (1979). Effects of estrogen upon the fine structure of epithelium and stroma in the rat ventral prostate gland. Invest. Ural. 17, 83-89. Turin, V., Senge, T., Schenk, B., and Neumann, F. (1979). Biochemical and histological studies on prostate in castrated dogs after treatment with androstanediol, oestradiol and cyproterone acetate. Acta Endocrinol. 91, 379-384.
AND
COMPARATIVE
Estradiol
ENDOCRINOLOGY
51,
Binding Activity
61-65 (1983)
in Epididymal
Chrysemys JEAN-PIERRE Department
DUFAURE,~
PAUL
Cytosol of the Turtle,
picta MAK, AND IAN P. CALLARD
of Biology, Boston University, Boston, Massachusetts 02215 Accepted August 5, 1982
Estrogen binding activity was investigated in the epididymis of the turtle, Chrysemys picta using DNA-cellulose affinity chromatography. A component binding estradiol-17P specifically with high affinity (&:8.0 x lo-lo M) and limited capacity (20 fmol/mg protein) was demonstrated in the epididymal cytosol. In addition, binding of estradiol-17P was sensitive to excess (loo-fold) diethylstilbestrol or natural estrogens (estradiol-17& estrone, and estriol) but not to progesterone or androgens (testosterone and Sa-dihydrotestosterone). The specific estrogen binding macromolecules eluted from DNA-cellulose columns sedimented at 4-5 S in linear 5-20% sucrose gradients. These characteristics suggest the presence of an estrogen receptor in this androgen target organ.
(Mak et al., 1982) of turtles from bath Androgen target tissues of the genital tract are influenced by estrogens in the dog sexes, and in the testis (Mak et al., submit(Huggins and Clark, 1940; Tunn et al., ted). The epididymis of the turtle shows 1979), rat (Feycl-Cabanes et al., 1977; well defined seasonal changes in weight and Thompson et al., 1979), calf (Kroes and histology (Callard, I. P., unpublished obTeppema, 1972), and guinea pig (Belis et servations) and in the lizard, Lacerta vivial., 1977). Specifically, estrogens can pro- para, binding of [H3]estradiol to epidihynial mote growth of the prostate and act prefer- cell nuclei has been demonstrated by autoentially on the fibromuscular stroma radiography (Dufaure et al., 1978). Th? purpose of this study was to advance our un(Feycl-Cabanes et al., 1977; Thompson et derstanding of the possible role of estrogen al., 1979). Consistent with these actions, cytoplasmic receptor proteins for estradiol in regulating seasonal breeding by identifyhave been described in prostate glands of ing a putative estrogen receptor in turtle the dog (Chairsiri et al., 1978; Robinette et epididymal cytosol. al., 1978; Dube et al., 1979), the rat (JungMATERIALS AND METHODS blut et al., 1971; Armstrong and Bashirelahi, 1974, 1978; Dubois et al., 1980; GinsSexually mature male Chrysemys picta were obburg et al., 1980), and some other species. tained from Nasco-Steinhilber, Inc. (For+ Atkinson, Wis.) from October through December and were mainAmong the Reptilia, estrogen receptor tained in the laboratory as previously described (Calproteins have been identified in estrogen lard et al., 1978). target tissues (oviduct) of the lizard (Botte Animals were killed by decapitation and all subseet al., 1974) and turtle (Salhanick et al., quent procedures were performed at 0-4°C. Tissues 1979). In addition, putative estrogen recep- were carefully washed in extraction buffer, minced, and homogenized in 4 vol (wtivol) buffer tors have been demonstrated in the liver weighed, with a glass-glass Potter Elvehjem homogenizer, $he (Heisermann et al., 1980) and the brain homogenate was ceritrifuged at 1400s for 20 Fin and cytdsol was prepared by centrifqation.of the r@lting supernate at 100,OOOgfor 1 hr. The protein concentration of cytosol was determined by ‘the method of Lowry et al., (19.51) as modified by Geiger and Beasman (1972) with BSA as the standard.
1 Present ‘address: Lab. Biologie Cellulaire et G&Ctique, Complexe Scientifique des Cezeaux, Universitb de Clermont-Fd 11, B.P. 45 63170 AubSre, France. 61
0016-6480183 $1.50 Copyright AU rights
@ 1983 by Academic Press, Inc. of reproduction in any form reserved.
62
DUFAURE,
L
I
2
I
4
6
I
1
6
No
MAK,
I
AND CALLARD (2-3 mg protein) were chromatographed onto columns by gravity and allowed to remain in contact with the DNA cellulose for 30 ruin at 4°C. The columns were then washed with 6-7 vol of buffer to remove any components not adhering to DNA cellulose and the labeled steroid dissolved in 50 m&’ NaCl buffer was subsequently loaded to the columns by gravity. For incubation, columns were sealed and kept at 23°C for 45 min. After returning to a 4°C cold room for 15 min, unbound steroid was removed by washing the columns with lo-12 vol of buffer containing 50 mM NaCl. DNA adhering components labeled with [3H]estradiol were eluted stepwise with buffer containing 400 mM NaCl and 1 mg BSA per ml. 0.5 ml fractions were collected and counted using a scintillation cocktail (750 ml Triton X-114, 2250 ml xylene, 9.0 g PPO, 0.75 g POPOP). Sucrose gradient centrifugation. Estrogen binding components eluted from DNA-cellulose columns were mixed with 5 ~1 [methyl-‘T] BSA and layered on 10 ml, 5-20% linear sucrose gradient prepared in TEGM buffer with 400 m&4 NaCl. Gradients were centrifuged at 240,OOOg for 20 hr (IEC centrifuge B-60, rotor SW283, at 4°C). Fractions (16 drops each) were collected from the bottom of the tube directly into counting
10 12 FRACTION
1. DNA-cellulose affinity chromatography of turtle epididymal cytosol. Samples (2.16 mg protein) were chromatographed onto 2 ml DNA-cellulose columns and incubated at 23°C for 45 mitt with 5 nit4 [3H]estradiol f loo-fold excess nonradioactive diethylstilbestrol (DES) as described under Methods. Bound radioactivity which adhered to the columns was eluted stepwise with TEMG buffer containing 400 n&f NaCl and 1.0 mg BSA per ml. Radioactivity was measured in 0.5 ml fractions after addition of 4.0 ml scintillation cocktail. 0, 5 nM [3H]estradiol; & 5 nM [3H]estradiol plus loo-fold excess nonradioactive DES. FIG.
Chemicals. Extraction buffer contained 10 n&4 Tris-HCl (pH 7.8), 1 mM Na, EDTA, 1 mM p-mercaptoethanol, 10% (vol/vol) glycerol (TEGM) and 50 mM NaCl. The molarity of NaCl was varied during chromatography from 50 to 400 mll/l; BSA (Pentex bovine albumin) from Miles Laboratory, Inc.: [2,4,6,73H]estradiol-17P (90- 110 Ci/mmol) and [r4C]BSA from New England Nuclear Corporation. Stock solutions of steroids were prepared in absolute ethanol and kept at -20°C. DNA-cellulose
afJinity chromatography. DNA-cellulose was prepared according to the method of Alberts and Herrick (1970) as modified by Fox and Pardee (1971). Briefly, 2 ml columns of DNA cellulose were equilibrated with 10 vol of buffer containing 50 mM NaCl and BSA (0.2 mgiml). Samples of cytosol
G +
FIG. 2. Competition analysis for estrogen binding sites. Equal volumes of cytosol(l.62 mg protein) were applied to DNA-cellulose columns and incubated with 3 nM [3H]estradiol -C lOO-fold excess different nonradioactive steroids. Other conditions were as in Fig. 1. The values of the different peaks of radioactivity corresponding to the binding complexes were calculated from the elution pattern and by subtracting the background. E, , estradiol-17p; E,, estrone; E,, estriol; DES, diethylstilbestrol; Pg, progesterone; T, testosterone; DHT, 5a dihydrotestosterone.
ESTRADIOL
BINDING
IN
TURTLE
63
EPIDIDYMIS
$:8x
IO-10 M
FIG. 3. Saturation analysis of estrogen binding in turtle epididymal cytosol using DNA-cellulose affinity chromatography. Samples of cytosol(2 mg protein) were chrumatographed onto DNA-celhtlose columns as described under Methods. Columns were incubated with [3H]estradiol from 0.5-5.0 r&f to determine total binding (A). Additional columns were incubated with 1.0,3.2, and 5.0niVQ3H]estradiol in the presence of IOO-fold excess nonradioactive dietbylstilbestrol (DES) to determine nanspecific binding (A). Specific binding (e) was obtained by subtracting nonspecific from total. Scatchard plot analysis of binding data revealed Kd: 8.0 x 10-W and m:.ximal binding sites: 20.6 fmok’mg protein.
vials. [‘*C]BSA (NEN) served as internal marker for each gradient.
RESULTS AND DISCUSSION
The data suggest the presence of a putative estrogen receptor in the cytosol of turtle epididymis. The elution pattern of DNAcellulose affinity chromatography for the estrogen binding component is illustrated in BSA
Top
5
IO NO
15
20
“&I
FRACTION
FIG. 4. Sedimentation analysis of estrogen binding components eluted from DNA cellulose. 0.8 ml of the 0.4 M NaCl DNA eluate was mixed with 5 ~1 l*C (Bovine Serum Albumin) and layered on a 10 ml, S-20% linear sucrose gradient as described under Methods. The arrow indicates the position of the internal marker [‘T]BSA (4.6 S).
Fig. 1. Such binding is specific, being displaced with a loo-fold excess of unlabeled competitor (diethylstilbestrol). The hormonal specificity was demonstrated by incubating the columns with r3HJestradiol (3 nM) plus lOO-fold excess of various unlabeled steroid competitors. As shown in Fig. 2, [3H]estradiol binding was efficiently displaced by unlabeled estradiol and diethylstilbestrol (DES) but not by testosterone, Sa-dihydrotesterone, or progesterone. Estrone and estriol were less effective than estradiol or DES. Tissue specificity was demonstrated in previous experiments which showed that bound radioactivity could not be detected in plasma, kidney, muscle, and lung (see Mak et al., 1982). Scatchard plot analysis of the binding data obtained after incubation with [3H&stradiol between 0.4 to 5.0 n&I L IOO-fold excess ‘DES gave values confirming high affinity (Kd: 8& x lo+’ M) and low capacity (20.6 fmolimg protein, Fig. 3). The estrogen binding macromolecules eluted from DNA celhuose had a sedimentation coefficient of 4,5 S
64
DUFAURE,
MAK,
(Fig. 4). Similar macromolecular components have also been demonstrated in the testicular cytosol of turtle (Mak et al., submitted). According to its binding affinity, specificity, and physicochemical properties, the estrogen binding activity of the turtle epididymis appears related to a receptor species rather than to a sex-steroid-binding protein originating from the plasma (Salhanick and Callard, 1980). Previous studies have demonstrated the androgen dependency of lizard epididymal growth and secretion (Gigon-Depeiges and Dufaure, 1977; Depeiges et al., 1982) and binding of labeled estradiol-17P to epididymal cell nuclei has been demonstrated in the same species (Lacerta vivipara) (Dufaure and Chambon, 1978). The exact function of estrogen receptors in the turtle epididymis is not known. However, preliminary data showed that receptor levels fluctuated during the annual reproductive cycle. Since turtle epididymis and plasma testosterone show a well defined seasonal cycle (Callard, I. P., unpublished observations), it. is possible that both estrogens and androgens play an important role in sperm maintenance or maturation in such seasonally breeding species. REFERENCES Alberts, B., and Herrick, G. (1970). DNA-cellulose chromatography. Meth. Enzymol. 21, 198-271. Armstrong, E. G., and Bashirelahi, N. (1974). A specific binding protein for 17P-estradiol in retired breeder rat ventral prostate. Biochem. Biophys. Res. Commun. 61, 628-634. Armstrong, E. G., and Bashirelahi, N. (1978). Determination of the binding properties of estradiol-17p within the cytoplasmic and nuclear fractions of rat ventral prostate. J. Steroid Biochem. 9, 507-513. Belis, J. A., Blume, C. D., and Mawhinney, M. G. (1977). Androgen and estrogen binding in male guinea-pig accessory sex organs. Endocrinology 101, 726-740. Botte, V., Granata, G., and Cristofaro, C. (1974). Interaction of 17P-estradiol and testosterone with the oviduct of the lizard Lacerta sicula. J. Steroid Biochem. 5, 687-691. Callard, I. P., Lance, V., Salhanick, A. R., and Barad, D. (1978). The annual ovarian cycle of Chrysemys
AND CALLARD Correlated changes in plasma steroids and parameters of vitellogenesis. Gen. Comp. Endo-
picta:
crinol.
35, 245-2.57.
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