Characterization of a specific estrogen receptor in the oviduct of the little skate, Raja erinacea

GENERAL

AND

COMPARATIVE

Characterization

ENDOCRINOLOGY

84, 170-181 (1991)

of a Specific Estrogen Receptor the Little Skate, Raja erinacea’

in the Oviduct

of

JOSEPH C. REESE AND IAN P. CALLARD Department of Biology, Boston University, Boston, Massachusetts 02215; and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672 Accepted December 19, 1990 In this study we report the first estrogen receptor to be characterized in an oviparous elasmobranch. The skate receptor has high aRinity for estradiol (K, = 0.7 nM), binds both estradiol and the synthetic estrogen DES, and exists in low quantities (XL100 fmol/g oviduct). The receptor displays rapid binding kinetics with half-times of 5 min at 22” and 77 min at 4”. DEAE-Sepharose chromatography reveals one receptor moiety which elutes between 0.13 and 0.14 M KCI. By sucrose gradient ultracentrifugation sedimentation coefftcients of 3.6 S under high-salt (0.5 M KCI) and 6.0 S under low salt (0.01 M KCI) conditions were obtained. Using Sephadex G200 gel filtration chromatography, a Stokes radius (R,) of 2.8 nm and an estimated molecular weight of 43 kDa were calculated. DNA-cellulose elution profiles reveal that the receptor elutes as one peak between 0.34 and 0.36 M NaCl (as compared to 0.20-0.22 M NaCl in mammals and birds and 0.55 M for dogfish). Although some differences are noted between the elasmobranch ER and those of other vertebrates (e.g., dissociation kinetics, DNA affinity), in general it can be said that the skate ER is a “classical” ER in most respects. It is suggested that this steroid receptor has played a key role in the reproductive tract functions of nutrient provision, embryo protection, and as a conduit to the external environment since the earliest chordate era, approximately 400 million years ago. 8 1991 Academic Press. Inc.

Considerable progress has been made in the characterization of gonadal steroid hormone receptors in poikilothermic species (see Callard and Callard, 1987, for review). Nevertheless, the elasmobranches, the most ancient gnathostome group, have received scant attention from endocrinologists despite presenting a wealth of sophisticated reproductive adaptations for the first time in vertebrates (Wourms, 1977; Dodd and Sumpter, 1984). Based on the work of a few laboratories (see Dodd and Sumpter, 1984; Koob et al., 1986; Callard and Klosterman, 1988; Callard, 1988; I. P. Callard et al., 1989; G. V. Callard et al., 1989), it can be said with certainty that elasmobranch reproductive physiology is under endocrine control. The little skate, Raja erinacea, is of particular interest as a potenl Supported by NSF DCB 8666344 to I.P.C.

tial model due to its ready availability, ease of maintenance in the laboratory, and adaptations for egg laying in the marine environment. This oviparous species encases its large-yolked eggs in an impervious structure, the well-known “mermaid’s purse,” which is formed during the periovulatory period (Koob et al., 1986) by the large shell gland. Although the secretion of shell gland proteins has received some attention (Rusauoen et al., 1976; Rusauoen, 1978; Koob and Cox, 1986; Cox and Koob, 1989), the endocrine control of this structure has not been investigated. However, a correlation between shell gland size and development, follicular size, and plasma estradiol levels has been shown (Koob et al., 1983; Koob et al., 1986). The intent of this paper is to establish the characteristics of the estrogen receptor in the oviduct of the little skate as a necessary preliminary to its 170

0016~6480/91 $1.50 Copyright Au rights

0 WY1 by Academic Press, Inc. of reproduction in any form reserved.

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further development as a model for steroid regulation. This is the first complete characterization of an estrogen receptor in the reproductive tract of a female oviparous elasmobranch although steroid binding has been previously reported in the oviduct of Squalus acanthius, and an estradiol receptor has been partially characterized in the testis of the same species(Cahard and Mak, 1985). MATERIALS

on ice. AU procedures were carried out at 0.4”. Tissue was processed as described by Riley et al. (1987). Briefly, tissue was minced, weighed, and homogenized in homogenizing buffer in a volume of 10: 1 (buffer:tissue). The homogenate was centrifuged at 15OOg for 5 mitt, the supematant was recovered and centrifuged as for nuclear extracts to yield cytosol, and the pellet was washed three times with nuclear pellet washing buffer and extracted for 1 hr in nuclear extraction buffer. This was then centrifuged at 100,000g to yield clear nuclear extracts. Concentration of protein was -8.0 mghl (cytosol) and -5.0 mg/ml (nuclear extract).

AND METHODS

Animals were maintained in pools of circulating seawater at the Mount Desert Island Biological Laboratories, Salsbury Cove, Maine, during June and July. All animals used were reproductively active with large, ovulatory-size follicles in the ovary (see Koob et al., 1986). Sacrifice was by cervical transection and the oviducts were removed and placed on ice until further treatment (see below).

Chemicals

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and Reagents

[2,4,6,7-3H]Estradiol-1713 (E,; sp act 90-102 Gil mmol) and bovine serum albumen (sp act 20 Ci/mg) were purchased from Amersham (Arlington Heights, IL). Radioinert steroids (Steraloids, Wilton, NH) were dissolved in ethanol and stored at - 20”. BSA, gel filtration molecular weight markers, No&-A charcoal, and Tris-HCl were from Sigma (St. Louis, MO). Sephadex LH-20, Sephadex G-200, and De.xtran T-70 were obtained from Pharmacia (Piscataway, NJ) and hydroxyapatite powder was from Bio-Rad (Richmond, CA). 2,5Diphenyloxazole (PPO) and p-bis-[2-(5 phenylozoyl)] benzene (POPGP) were obtained from Research Products International (Mt. Prospect, IL). All other chemicals used were of reagent grade. Buffers used were as follows: TEMG (10 mM Trisbase, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 10% (v/v) glycerol, pH 7.5), homogenizing buffer (50 mM Tris-HCl, 1 mM EDTA, 12 mM monothioglycerol, 30% (v/v) glycerol, pH 7.5), nuclear extraction buffer (0.7 M KC1 in homogenizing buffer) according to Chen and Leavitt (1979), nuclear pellet washing buffer (10 m&f Tris-base, 3 n&f MgCl,, 12 mM monothioglycerol, 0.25 M sucrose, pH 7.5). Buffers used in DNA-cellulose chromatography were TEMG plus 200 mgihter BSA containing 0.5 M (Buffer A) and 0.8 M (Buffer B) KCl, pH 7.5. Dextran-coated charcoal solution (DCC) was 0.5% Norit-A charcoal and 0.05% Dextran T-70 in TEMG.

Preparation of Cytosols and Nuclear Extracts Oviducts were rinsed in ice-cold TEMG and placed

Estradiol

Binding

Assays

Estradiol binding was assayed by two methods. A hydroxyapatite (HAP) assay as modified by Pavlik (Pavlik and Coulson, 1976) was used. In brief, 0.22 ml of a HAP slurry (SO/SO,v/v, HAP/TEMG) was added to 0.4 ml of sample and incubated on ice for 30 min. Incubation was terminated by centrifugation at 15OOg for 5 min. The HAP pellet was washed three times with TEMG and the radioactivity extracted with 1.Oml of ethanol. For time-dependent studies a more rapid Sephadex LH-20 adsorption chromatography assay was used and performed as described (Mak ef al., 1982). Briefly, LH-20 was swollen in distilled water and packed into acid-washed columns (0.5 x 5.0 cm) and equilibrated with 5 vol of 0.05 M KC1 in TEMG for cytosolic and 0.5 M KC1 in TEMG for nuclear extract samples. Sample (0.4 ml) was allowed to adsorb onto the column, and 0.2 ml of buffer was then added. Buffer (1 .O ml) was then added and collected for scintillation counting. When using LH-20, 1 @+4 DOC was added to eliminate any interference by a serum binding protein that was not detected by using the HAP assay.

t3HjE Binding in Cytosols and Nuclear Extracts Cytosols were diluted 1: 1 with homogenizing buffer and nuclear extracts were diluted 1:l in nuclear extraction buffer to give a final protein concentration of 3-5 and 2-4 mg/ml, respectively. For association time studies, diluted samples (0.4 ml) were incubated with 5 nM [3H]E, and 1 t&f desoxycorticosterone (DOC) plus (nonspecific binding) and minus (total binding) 200-fold radioinert E2 at 4” for 4-24 hr and at 22” for 15 min-24 hr. Data were expressed as specific counts per minute ([-‘H]E,) which was calculated by subtracting nonspecific binding from total binding. Bound and free steroid were separated by LH-20 chromatography. Dissociation kinetics were studied at 4” by incubating diluted cytosol and nuclear extracts with 5 nM [3H]E2 and 1 @t4 DGC to equilibrium at 4” (U-16 hr). Aliquots (0.4 ml) were transferred to tubes containing

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200-fold excess radioinert E, and incubated on ice for O-150 min. Dissociation was studied at 22” by incubating diluted nuclear extracts with 5 nM [3H]E, and 1 p,M DOC to equilibrium at 22”. Subsequently, aliquots (0.4 ml) were added to tubes containing 200-fold radioinert E,, followed by incubation at 22” for O-10 min. Incubations were terminated by applying samples to LH-20 columns equilibrated at 4”. Data were expressed as a percentage of specific binding determined at Time 0. Saturation and Scatchard analysis were performed on diluted samples using the hydroxyapatite assay. Diluted nuclear extracts and cytosols were incubated with increasing concentrations of [3H]E2 (0.5-5 nM) plus (nonspecific binding) and minus (total binding) 200-fold radioinert E, to equilibrium at 4”. Bound and free were separated by HAP assay and specific binding calculated (T - NS = S).

Specificity

of [3HjE2 Binding

Diluted sample (0.4 ml) was incubated with 5 nM [3H]E, plus and minus I-, lo-, and lOO-fold radioinert competitors and incubated at 4” for 16 hr. Incubation was terminated by addition of a HAP slurry and the HAP assay performed as described above. Data were expressed as a percentage of specific [3H]E2 bound in tubes without competitors vs tubes containing competitors.

DEAE-Sepharose

Analysis

DEAE-Sepharose was packed into acid-washed columns (5 x 15 mm) and equilibrated at 4” with 0.05 M KCI in TEMG. Cytosol and nuclear extracts were incubated to equilibrium at 4” with 5 nil4 [3H]E2 and 1 phf DGC plus and minus 200-fold radioinert E,. Cytosols were directly applied to the column while nuclear extracts were diluted 1:15 with 0.05 M TEMG after incubation and applied to the column. The column was washed with 0.05 M KC1 TEMG until the radioactivity in each fraction was low, after which the column was eluted with a linear KC1 gradient in TEMG (0.05-0.05 M). Fractions (0.5 ml) were collected, salt was measured with a conductivity meter, and radioactivity was determined by scintillation counting.

DNA-Cellulose

Chromatography

DNA-cellulose was prepared according to Alberts and Herrick (1970) and chromatography performed as described by Salhanick et al. (1979). In short, 2-ml DNA-cellulose columns were equilibrated with DNAcellulose buffer A at 4”. One milliliter of cytosol or 1 .O ml of nuclear extracts desalted on LH-20 columns were incubated with 5 nM f3H]E2 plus and minus 200-

CALLARD

fold radioinert E, to equilibrium at 4”. The samples were then applied to and allowed to enter the columns and incubated at 22” for 30 min. Columns were returned to 4” for 30 mitt, then washed with buffer A to remove the free steroids, and eluted with a linear NaCl gradient (0.05-0.8 M) using buffers A and B. Onemilliliter fractions were collected and salt concentrations were determined using a conductivity meter and radioactivity by scintillation counting.

Sucrose Gradient

Ultracentrifugation

Linear sucrose gradients were prepared using a gradient former (Buchler, Saddle Brook, NJ). Gradients (4 ml) of 5-20% sucrose in TEMG containing either 0.01 M KC1 (low-salt gradients) or 0.5 M KC1 (high salt gradients) were prepared and kept overnight at 4”. Cytosols and nuclear extracts were incubated overnight at 4” with 5 nM [3H]E2 and 1 pM DGC plus or minus radioinert E,. Free steroid was removed by adding samples to a Dextran-coated charcoal pellet, vortexing, incubating on ice for 5 min, and centrifuging at 15OOg. after which the supematants were recovered. Cytosol (0.4 ml) was layered onto low-salt gradients, and 0.4 ml of nuclear extract was layered onto the high-salt gradients. [14C]BSA (3.0 ~1) was added as an internal standard. Gradients were spun at 50 K for 4.5 hr in a vertical rotor (TV865B in Sorvall OTD65, 219,OOOg) at 4”. Gradients were then collected (5 drops) from the top using a Buchler gradient collector. Radioactivity was counted for [3H] and [14C] using a scintillation counter.

Sephadex G-200 Gel Filtration Gel filtration of nuclear extracts and cytosols was performed. A 2 x 75-cm G-200 column was poured at 4” and equilibrated with 0.05 M TEMG. The column was calibrated with cytochrome C, carbonic anhydrase, ovalbumin, bovine serum albumin, alcohol dehydrogenase, and B-amylase. Void volume was determined by blue dextran. A graph of Stokes radius (R,) vs VJV,, was used to estimate R, for the receptor. An estimation of the molecular weight was calculated by sedimentation coefficient x R, x 4224 according to Sherman (Sherman et al., 1983). Samples were incubated with 5 nM [‘H]E, and 1 ~.LMDOC to equilibrium at 4”. Free steroid was removed with a DCC pellet as described. Samples (0.75 ml) were then added to the column and 1.O-ml fractions were collected at a flow of 18 mhhr. Radioactivity of each fraction following the void volume was determined by scintillation counting.

Scintillation

Counting

Four milliliters of scintillation fluid (2.25 liters of xylene, 0.75 liters of Triton X-114, 9 g PPO, and 0.75

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g POPOP) were added to samples, shaken, allowed to settle overnight, and counted in a Tracer 300 counter with an efftciency of 45% for most samples.

L5zT RESULTS

Association and Dissociation Kinetics of [3HjE2 Binding in Cytosols and Nuclear Extracts (Fig. Z) Association kinetics. Association kinetics were studied at 22” (Fig. 1) and at 4” using diluted nuclear extracts. Nuclear extracts were incubated with 5 ti [3H]E2 and 1 PM DOC plus and minus 200-fold radioinert E2 for 10 min-4 hr at 22” and for 4-24 hr at 4” (data not shown). Incubation was terminated by application to LH-20 columns at 4”. At 22” specific [3H]E2 binding reached equilibrium between 45 and 60 min and remained stable at 4 hr. At 4” specific [3H]E2 binding reached a maximum between 12 and 14 hr and was stable at 24 hr. Dissociation kinetics. Nuclear extracts were incubated at both 22” (Fig. 2) and 4”

F M I !: g 500 P ma 1

‘---/I

0

10

20

30

40

Time

50

[min.]

50

70

4hrs.

FIG. 1. Association was studied at 22” and 4” (data not shown). Diluted nuclear extracts were incubated in the presence of 5 r&f [3H]Ez and 1@+4 DOC + 200-fold radioinert E, for 10 min to 4 hr at 22”. Incubations in both cases were terminated by applying samples to LH-20 columns as described under Materials and Methods. Data are expressed as specific bound [3H]E2. Equilibrium occurred between 45 and 60 min at 22” and between 12 and 14 hr at 4”.

I 0

I

I 5

Time

I 15

[min.y

FIG. 2. Dissociation of 13H]E,-receptor complexes was studied at 22” and 4” (data not shown). Diluted nuclear extracts were incubated to equilibrium at 22” with 5 nM t3H1E, and 1 JLM WC, and then aliquots (0.4 ml) were added to tubes containing 200-fold radioinert E, and incubated for 2 to 10 min at the same temperature. Aliquots (0.4 ml) were then added to tubes containing 200-fold radioinert E2 and incubated for 15 to 150 min. Free steroid was removed by LH-20 chromatography as described under Materials and Methods. The graph shows that the receptor has a half-time of 5 min at 22”.

and cytosols at 4” only (data not shown). Samples were incubated to equilibrium at the appropriate temperature with 5 nM 13HJE2 and 1 J.& DOC. Aliquots were then transferred to tubes containing 200-fold radioinert E, and allowed to incubate at the appropriate temperature and time as described under Materials and Methods. Incubation was terminated by LH-20 chromatography. At room temperature specific [3H]E2 binding reached half-maximum at 5 +- 0.5 min (n = 5). At 4” results for both the cytosolic (n = 3) and nuclear samples (n = 3) were similar with half-maximum binding occurring at 77 -+ 1 min (data not shown). Specificity

of [3HjE2 Binding

(Fig. 3)

Specificity of [3H]& binding was examined in nuclear extracts by competition

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REESE AND CALLARD

analysis. Diluted samples were incubated overnight at 4” with 5 nM [3H]E2 plus or minus increasing concentrations of competitors (l-, lo-, and lOO-fold) and data expressed as a percentage of specific binding. Both estradiol and the synthetic estrogen (DES) competed very well at all concentrations while dihydrotestosterone, estrone, testosterone, estriol, progesterone, and deoxycorticosterone (DOC) competed very poorly, even at higher concentrations. Specificity curves suggest that the receptor binds estradiol tighter than the synthetic estrogen DES. Saturation and Scatchard Analysis of Cytosol and Nuclear Extracts (Fig. 4) Saturation and Scatchard analysis were performed on cytosols (n = 2) and nuclear extracts (n = 3) with similar results. Samples were incubated at 4” overnight with increasing concentrations of [3H]E2 plus and minus 200-fold radioinert E,. Free steroid

was removed by HAP assay. Saturation analysis (Fig. 4A) revealed that binding saturates between 4.0 and 5.0 n&f [3H]E2. Scatchard analysis (Fig. 4B) revealed one binding species with a Kd of 7 x IO- lo M (0.70 k 0.12 nM, 8 S) and a B,, between 1.6 and 3.5 X 10-l’ M(1.86 x lo-” 2 0.17 x 10-l’ kf). The average r value for the linear regression used in the Scatchard plots was 0.94 k 0.017. Binding was low (254 fmol/g tissue) in both cytosolic and nuclear fractions. DEAE-Sepharose Not Shown)

Chromatography

(Data

DEAE-Sepharose chromatography was performed on both cytosols and nuclear extracts. Undiluted sample (0.5 ml) was incubated overnight at 4” with 5 ti [3H]E2 and 1 ).&I DOC plus or minus 200-fold radioinert E. Cytosols were added directly to the column while nuclear extracts were diluted 1: 10 with TEMG. Columns were washed and eluted as described under Materials

25-

0

1x Competitor

Concentration

10x [Fold

100x XS]

FIG. 3. Specificity of [‘H]E, binding was studied by competition analysis for [‘H]E, binding sites by radioinert steroids. Diluted nuclear extracts were incubated with 5 nM [‘H]E, plus and minus I-, lo-, and RIO-fold radioinert competitors to equilibrium at 4”. Free steroid was removed by HAP assay as described under Materials and Methods. Data are expressed as a percentage of specific [3H]E2 binding. Data reveal that the receptor is specific for both estrogen and the synthetic estrogen DES by binding E, better than DES. Other steroids such as testosterone, dihydrotestosterone, and progesterone competed poorly at all concentrations.

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RECEPTOR

175

FIG. 4. Saturation and Scatchard analysis were performed on nuclear extracts. Diluted samples were incubated with increasing concentrations of [3H]E2 (0.5-5 nM) in the presence of 200-fold radioinert E, (0) and in the absence of E, (H). Free steroid was removed by HAP assay. Specific binding was calculated by subtracting binding in incubations with E, from incubations without. The receptor species saturates between 4 and 5 nM [3H]E2. Scatchard analysis reveals that the receptor binds [3H]E, with high affinity, displaying a 4 of 7 x lo-r0 M, and limited capacity B,, of 1.67 X lo-” M. (0) Nonspecific binding.

and Methods. In both cases one specific peak which eluted between 0.13 and 0.14 M KC1 was observed. DNA-Cellulose

chromatography

(Fig. 5)

DNA-cellulose chromatography was performed on cytosols and desalted nuclear extracts. Samples (1.0 ml) were incubated overnight at 4 C with 5 nM [3H]E2 plus or minus radioinert EZ, applied to the column, incubated at 22” for 30 mitt, returned to 4” for 30 more min, and washed and eluted as described under Materials and Methods. Elution profiles of both cytosols and nuclear extracts reveal one specific peak eluting between 0.34 and 0.36 M NaCl. Sucrose Gradient (Fig. 6)

Ultracentrifugation

Sedimentation properties of the skate estradiol receptor were examined in cytosols under low-salt conditions (data not shown) and in nuclear extracts under high-salt conditions (Fig. 7). Samples were incubated with 5 ti [3H]E2 and 1 t.& DOC plus and minus 200-fold excess E, overnight at 4”

and free steroid was removed by DCC pellet, layered onto gradients, and centrifuged. Low-salt (0.01 M KCl) gradients reveal that the receptor sediments as a 6.0 S complex. In high-salt (0.5 M KCl) gradients the receptor sediments as the lighter 3.5 S species. Sephadex G-200 Filtration and Molecular Weight Estimation (Fig. 7) The size of the skate estradiol receptor was examined by gel filtration chromatography of cytosols and nuclear extracts. Nuclear extracts and cytosols were incubated at 22” for 1 hr and then cooled on ice for 30 min. Free steroid was removed with a DCC pellet and 0.75 ml of sample applied to a G-200 column calibrated with cytochrome C , carbonic anhydrase, ovalbumin, bovine serum albumin, alcohol dehydrogenase, and P-amylase. The column was eluted and radioactivity in each fraction measured. One specific sharp peak eluting near ovalbumin was observed and a Stokes radii (Z?,) vs VJV, graph was used to calculate a R, for the receptor of 2.8 nm (n = 3). Molec-

176

REESE AND CALLARD 800

.7 -

600

.A

E

.A-,

8 A

,I

.6 .5

,A,.’

.4 .3 .2 .1

lb

0

Fraction

2b

20

Number

FIG. 5. DNA binding properties of the skate estrogen receptor examined by DNA-cellulose chromatography. Samples were incubated with 5 nit4 [3H]E2 at 4” plus (0) and minus (0) 200-fold radioinert E2 at 4”. Samples were applied to DNA-cellulose columns and incubated at 22” for 30 min and then at 4” for 30 more min. Free steroid was washed away with low-salt buffer, and then the columns were eluted with a linear NaCl gradient (A). Fractions were collected and radioactivity and salt concentrations determined as described under Materials and Methods. One specific peak eluting between 0.35 and 0.36 M was observed.

0,0. 17 l -... l I

I

I

10

20

30

Fraction Number FIG. 6. Sedimentation properties of the receptor were studied under both high-salt (0.5 M KCI) and low-salt (0.01 M KCI) conditions. Nuclear extracts (high-salt) were incubated to equilibrium at 4” in the presence of 5 t&f [3H]E 1 p&I DOC plus (0) and minus (0) 200-fold radioinert E2. Free steroid was removed by a DCC pellet as described under Materials and Methods. Nuclear samples were layered onto high-salt and cytosols onto low-salt linear 5-20% sucrose gradients. C-14 BSA (4.65 S) was added to each tube as an internal standard, and its position in the gradients is marked by an arrow. The skate receptor sediments as a 3.5 S species under high-salt conditions and as a 6.0 S species under low-salt conditions (data not shown).

SKATE UTERINE

ESTROGEN

1

I

T

1

25

50

75

100

Fraction

177

RECEPTOR

I

125

Number

FIG. 7. The size of the skate estadiol receptor was estimated by Sephadex G-200 gel filtration. Samples were incubated with 5 nM [‘HIE, 1 pM DOC plus (0) and minus (0) 200-fold radioinert E,. Free steroid was removed by a DCC pellet as previously described. Column was eluted, fractions were collected, radioactivity was determined. Stokes radius and molecular weight were estimated as described under Materials and Methods. Column was calibrated with protein standards indicated above the graph. V, void volume; 1, Ramylase; 2, alcohol dehydrogenase; 3, bovine serum albumin; 4, ovalbumin; 5, cytochrome c. One specific peak eluting at the VJV, similar to ovalbumin was observed. A stokes radius of 2.2 nm was calculated for the receptor and an estimated molecular weight was determined to be 42,000.

ular weight estimation coupled with sedimentation data reveal a molecular weight of about 43 kDa for the skate estradiol receptor. DISCUSSION

Here we report the thorough characterization of the first estrogen receptor in the oviduct of an oviparous elasmobranch, the little skate, R. erinacea. The ER was found in both cytosolic and nuclear fractions, as observed for S. acanthias oviduct (Callard and Mak, 1985), but unlike the Squalus testis ER which was exclusively nuclear in location (Callard and Mak, 1985). Association kinetics were similar to those of homeothermic and other poikilothermic species (Puca et al., 1971; Notides and Nielsen, 1974; Zava and McGuire, 1977; Salhanick et al., 1979; Mak et al., 1982; Okulicz ef al., 1983; McNaught and Smith, 1986). Binding was maximal at 45 min at 22”

and between 12 and 14 hr at 4“. These times are equal to those found for the pituitary ER of the European dogfish, Scyliorhinus canicula (Jenkins et al., 1980) and the ER of Squalus testis which equilibrate between 30 and 45 min at 22” (Callard and Mak, 1985). In contrast, dissociation kinetics were much more rapid than in homeotherms at 4” (Z’,,,: 77 min in the skate vs 250 hr for the hamster uterus (Okulicz et al., 1983) and 16-20 hr for the rat (Best-Belpomme et al., 1970). However, dissociation times are rapid in other poikilothermic species. For example, the Xenopus liver ER has a T,,, of 210 min at 4” (Notides and Nielsen, 1974), and the value for salmon liver ER is 117 min (Lazier et al., 1985). At 22” the T,,* for the skate is fast (5 min), similar to Xenopus liver ER (13 min; Notides and Nielsen, 1974). These times are much less than reported for mammals at this temperature (of the order of l-2 hr, see Alberts and Her-

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rick, 1970; Best-Belpomme et al., 1970; Puca et al., 1971; Okulicz et al., 1983). TII1 for the ER of the snake liver is considerably faster at both temperatures (22”, 1.0 min; 4”, 11 min; Riley and Callard, 1988). It is worth noting that the dissociation rate for an elasmobranch binding protein in S. acanthias (Ho et al., 1980) is somewhat slower (T,,* = 100 min at 4”) than that of the estradiol receptor described here. These data suggest that hormone receptor complexes are less stable in species that normally operate over a wide range of body temperatures and therefore pose questions for hormone action under such circumstances. Under physiological circumstances the higher circulating levels of plasma steroids in nonmammalian species (Salhanick and Callard, 1979) may play an important role in the maintenance of the stability of the hormone-receptor complex, thus ensuring proper genomic effects under low ambient temperatures. The skate ER is specific for E, and DES, but unlike most mammalian ER (Eisenfeld et al., 1975; Danzo et al., 1977; Duffy and Duffy, 1978; Okulicz et al., 1983) has greater affinity for E, than DES, while estrone, estriol, testosterone, DHT, and P competed poorly even at high concentrations. Skate ER is present in low abundance compared to avian and mammalian species (Alberts and Herrick, 1970; Puca et al., 1971; Notides and Nielsen, 1974; Zava and McGuire, 1977; Okulicz et al., 1983; McNaught and Smith, 1986) levels ranging from 50 to 120 fmol/g, or l&100 times less than reported for homeotherms. In contrast, ER levels reported for reptiles, amphibia, and fish (Notides and Nielsen, 1974; Salhanick et al., 1979; Mak et al., 1982; Callard and Mak, 1985; Lazier et al., 1985; Riley et al., 1987; Riley and Callard, 1988) are of the same order, and skate values are the same as those reported for Squalus testis (77 fmol/g) and oviduct (135 fmol/g). Like other vertebrate ER, affinity for E, (Kd 7 x 10-l’) is high (Notides and

CALLARD

Nielsen, 1974; Danzo et al., 1977; Duffy and Dutfy, 1978; Mak et al., 1982; Callard and Mak, 1985; Lazier et al., 1985; McNaught and Smith, 1986; Riley and Callard, 1988), saturating between 4 and 5 ti. Unlike the rat uterus (Markaverich et al., 1986) and chick oviduct (McNaught and Smith, 1986), skate ER does not display complex binding kinetics. DNA cellulose elution profiles revealed that skate ER binds to calf thymus DNA tighter than either avian or mammalian ER which normally elute between 0.18 and 0.22 M NaCl (Alberts and Herrick, 1970; McNaught and Smith, 1986) as compared to 0.35 to 0.36 in the skate. The Squalus testicular ER binds DNA-cellulose more tightly than the skate, eluting at 0.55 M NaCl (Callard and Mak, 1985). These workers have suggested that the high affinity for DNA exhibited by marine elasmobranchs such as Squalus (and skate) may be an adaptation to high body fluid osmolarity (1010 mosM) of these species. This interpretation is supported by the salt elution maximum (0.22 M NaCl) for the liver ER of a freshwater elasmobranch (Potamotrygon) which does not retain urea and has a body fluid osmolarity (333 mosM) similar to that of most mammals. The salt elution profile for the skate oviduct ER (0.35 M NaCl) is intermediate between that for Squalus and that reported for most other species (i.e., 0.2 M NaCI). Another observation which supports a relationship between body fluid osmolarity and DNA aftinity of ER is that in Necturus (Mak et al., 1983), with a body fluid osmolarity of about 200 mosM, the ER elutes from DNA at 0.15 M NaCI. Studies of receptor elution profiles in more species with different body fluid osmolarity values would be valuable in a determination of the physiologic importance of the apparent different DNA affinities of ER. In sucrose gradients the skate ER sediments at 3.6 S in high-salt and 6.0 S in lowsalt gradients. This is in contrast to most mammalian and Squalus testicular ER

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which sediment between 4 and 5 S in highsalt gradients (Puca et al., 1971; Notides and Nielsen, 1974; Salhanick et al., 1979; Jenkins et al., 1980; Mak et al., 1982; Callard and Mak, 1985; McNaught and Smith, 1986), but similar to that of snake (Riley and Callard, 1988), Xenopus (Notides and Nielsen, 1974), salmon liver (Lazier et al., 1985), and chick oviduct (McNaught and Smith, 1986). The skate ER sedimentation rate in low-salt gradients (6 S) is similar to that of the snake (Riley and Callard, 1988) and Xenopus liver (Notides and Nielsen, 1974). This 6 S form may be homologous with the 5.5 -5.8 S form of the mammalian ER observed in low-salt gradients and is possibly an activated dimer of two receptor molecules (Notides and Nielsen, 1974; Jasper et al., 1985; Redleuilh et al., 1987). The heavier 8-9 S form of the receptor which is found in mammals and birds (Redleuilh et al., 1987; Sabbah et al., 1987) was not observed in the skate. The molecular weight (M, 43,000) of the skate ER was estimated from the Stokes radius (R, = 2.8 nm by gel filtration) and the sedimentation coefficient. This compares to the molecular weight of the Xenopus liver ER (40,000) calculated from an R, of 2.6 nm (Notides and Nielsen, 1974). The R, of the rat uterine ER (4.4 nm) and 4 S value yields a molecular weight of 76,000 (Notides and Nielsen, 1974) compared to the ER from the calf uterus (R,, 3.3 nm) and a molecular weight of 61,000 (Puca et al., 1971). However, despite these apparent differences, recent studies using the ER aflinity label tamoxifen aziridine (Monsma et al., 1984) and cloned genes of the human (Green et al., 1987), chicken (Maxwell et al., 1987), Xenopus (Weiler et al., 1987) and mouse (White et al., 1987) reveal that these ER have molecular weights of 66,000, despite differences in R,, sedimentation coefficient, and calculated molecular weights. It is likely that the skate ER may also have a molecular weight of 66,000, in which case the receptor may be similar in size to those

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of other vertebrates. The charge on the skate ER was determined by ion-exchange chromatography on DEAE-Sepharose and elution profiles which revealed a single peak eluting between 0.13 and 0.14 M KC1 for both cytosolic and nuclear receptors. Elution at these concentrations suggests that the skate ER is less acidic than that of the chicken (0.16-0.20 M KCl; McNaught and Smith, 1986) and the rat ER (elution between 0.18 and 0.2 M KCl; Jasper et al., 1985).

Although no data have been presented on the regulation of the skate oviduct ER and its function, it can be anticipated that studies in progress in our laboratory will establish these aspects in relationship to the physiology of the species. In particular, study of the role of estrogen in the synthesis of complex shell gland proteins should provide an excellent new model for the genomic actions of estrogen. Our previous studies (Koob et al., 1983; Koob et al., 1986) of the correlation between the shell gland growth and development and plasma estradiol levels suggest an important role for this hormone. Although some differences between the elasmobranch ER and those of other vertebrates are noted here (dissociation kinetics, DNA affinity), in general it can be said that the skate ER is a “classical” ER in most respects and suggests that this protein has been involved in the reproductive tract functions of providing nutrients, protection, and a conduit to the external environment from the earliest chordate era approximately 400 million years ago. REFERENCES Alberts, B. M., and Herrick, G. (1970). DNAcellulose chromatography. In “Methods in Enzymology” (S. Grossman and K. Moldave, Eds.), Vol. 21, pp. 198-217. Academic Press, New York. Best-Belpomme, M., Fries, J., and Erdos, T. (1970). Interactions entre l’oestradiol et des sites r& cepteurs u&ins. Eur. .I. Biochem. 17, 425-434. Callard, G. V. (1988). Reproductive physiology: Part

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B. The male. In “Physiology of Elasmobranch Fishes.” (T. Shuttleworth, Ed.), pp. 292-312. Springer-Verlag, New York/Berlin. Callard, G. V., and Mak, P. (1985). Exclusive nuclear localization of estrogen receptor in Squalus testis. Proc. Natl. Acad. Sci. USA 82, 133f%1340. Callard, G. V., Mak, P., Dubois, W., and Cuevas, M. (1989). Regulation of spermatogenesis: The shark testis model. J. Exp. Zool. (Suppf) 2, 23. Callard, I. P., and Callard, G. V. (1987). Steroid hormone receptors and non-receptor binding proteins. In “Hormones and Reproduction in Fishes, Amphibians and Reptiles” (D. Norris and R. E. Jones, Eds.), pp. 355-384. Plenum, New York. Callard, I. P., and Klosterman, L. L. (1988). Reproductive physiology: Part A. The female. In “Physiology of Elasmobranch Fishes” (T. Shuttleworth, Ed.), pp. 277-292. Springer-Verlag, New York/Berlin. Callard, I. P., Klosterman, L. L., Sorbera, L. A., Fileti, L. A., and Reese, J. C. (1989). Endocrine regulation in elasmobranchs: Archetype for terrestrial vertebrates. .I. Exp. Zool. (Suppl.) 2, 1222.

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Markaverich, B. M., Roberts, R. R., Alejandro, M. A., and Clark, J. H. (1986). Uterine type II estrogen-binding sites are not of eosinophil origin. J. Biol. Chem. 261, 142-146. Maxwell, B. L., McDonnell, D. P., Conneely, 0. M., Schulz, T. Z., Greene, G. L., and O’Malley, B. W. (1987). Structural organization and regulation of the chicken estrogen receptor. Mol. Endocrinol. 1, 25-35. McNaught, R. W., and Smith, R. G. (1986). Characterization of a second estrogen receptor species in chick oviduct. Biochemistry 25, 2073-2081. Monsma, F. J., Katzenellenbogan, B. S., Miller, M. A., Ziegler, Y. S., and Katzenellenbogan, J. A. (1984). Characterization of the estrogen receptor and its dynamics in MCF7 human breast cancer cells using a covalently attacking antiestrogen. Endocrinology 115, 143-153. Notides, A. C., and Nielsen, S. (1974). The molecular mechanism of the in vitro 4s and 5s transformation of the uterine estrogen receptor. J. Biol. Chem. 249, 1866-1873. Okulicz, W. C., Boomsa, R. A., MacDonald, R. G., and Leavitt, W. W. (1983). Conditions for the

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measurement of estrogen receptor at low temperature. Biochim. Biophys. Acia 157, 128-136. Pavlik, E. J., and Coulson, P. B. (1976). Hydroxyapatite batch assay for estrogen receptors: Increased sensitivity over present receptor assay. J. Steroid Biochem. 7, 357-368. Puca, G. A., Nola, E., Sica, V., and Bresciani, F. (1971). Estrogen-binding proteins of calf uterus. Partial purification and preliminary characterization of two cytosolic proteins. Biochemistry 10, 3767. Redeuilh, G., Moncharmont, B., Secco, C., and Beaulieu, E.-E. (1987). Subunit composition of the molybdate-stabilised 8-9s nontransformed estradiol receptor purified from calf uterus. J. Biol. Chem. 262, 6969-6975. Riley, D. and Callard, I. P. (1988). An estrogen receptor in the liver of the viviparous water snake, Nerodia: Characterization and seasonal changes in binding capacity. Endocrinology 123, 753-161. Riley, D., Heisermann, G. J., MacPherson, R. and Callard, I. P. (1987). Hepatic estrogen receptor in the turtle, Chrysemys picta: Partial characterization, seasonal changes and pituitary dependence. .I. Steroid Biochem. 26, 4147. Rusaouen, M. (1978). Etude ultrastructurale des zones a secretions proteiques et glycoproteiques de la glande nidamentaire de la rousette, a maturite. Arch. Anat. Microsc. 67, 107-l 19. Rusaouen, M., Pujol, J. P., Bocquet, J., Veillard, A., Borel, J. P. (1976). Evidence of collagen in the egg capsule of the dogfish, Scyliorhinus canicula. Comp. Biochem. Physiol. B 53, 539-543. Sabbah, M., Redeuilh, G., Secco, C., and Beaulieu, E.-E. (1987). The binding activity of estrogen receptor to DNA and heat shock protein (M, 90,000)

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