0013-7227/90/1262-1133$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Regulation and Localization of Estrogen and Progestin Receptors in the Pituitary of Steroid-Treated Monkeys* S. A. SPRANGERS, N. B. WEST, R. M. BRENNER, AND C. L. BETHEA Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and the Departments of Physiology, and Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201
ABSTRACT. PRL increases during pregnancy in primates with rising levels of placental estradiol (E) and progesterone (P). However, while E will increase PRL secretion in monkey pituitary cell cultures, P has no effect. We recently localized progestin receptors (PR) to gonadotropes, but not lactotropes, with an immunocytochemical technique to double stain monkey pituitary cell cultures. The following studies were performed to confirm the immunocytochemical localization of PR in intact pituitary tissue and to determine the effect of E and P on the levels of estrogen receptors (ER) and PR in the pituitary. ER and PR levels were determined in the endometrium of the same animals for an internal comparison. Thirteen adult cycling female cynomolgus monkeys were ovariectomized and treated for 28 days with 1) an empty Silastic capsule (Spay), 2) a 2-cm Efilled capsule (E), or 3) a 2-cm E-filled capsule for 14 days plus a 6-cm P-filled capsule implanted for an additional 14 days (E+P). Blood samples were drawn daily for assay of serum E, P, and PRL levels. Serum PRL was not significantly affected by E, but the sequential addition of P significantly increased serum PRL levels over those observed in Spay animals. The anterior pituitary and endometrium were removed for measurement of
ER and PR levels by a sucrose gradient shift assay incorporating monoclonal antibodies against ER and PR. Pituitary ER levels did not vary significantly with steroid treatment (158.2 ± 33.6, 135.5 ± 24.9, 104.3 ± 13.4 fmol/mg DNA in Spay, E, and E+P animals, respectively). Pituitary PR levels were undetectable in Spay animals, were induced by E (393.3 ± 53.4 fmol/mg DNA), and were suppressed to undetectable levels by the addition of P. A portion of the pituitary was frozen for immunocytochemical single staining for ER, PR, PRL, and LH and double staining for PRL+PR and LH+PR. ER staining was observed in many parenchymal cells, but there was no apparent change with steroid treatment. PR staining was absent in the Spay animals; many PR-positive cells were observed in E-treated females, and only a small number of faintly staining cells were detected in the E+P animals. Double staining for PRL+PR and LH+PR revealed PR in gonadotropes, but not lactotropes. In conclusion, PR, but not ER, are regulated by E and P in the monkey pituitary. Importantly, PR is regulated within gonadotropes, but not lactotropes. Therefore, P probably increases PRL secretion through a hypothalamic action. (Endocrinology 126:1133-1142, 1990)
T N PRIMATES, serum PRL rises late in gestation JL coincident with rising estradiol (E) and progesterone (P) levels from placental sources (1, 2). E is a potent in uiuo stimulus for PRL secretion in rats (3-5), but E is less effective in stimulating PRL secretion in monkeys unless hypothalamic inhibition is first removed (6, 7). This laboratory has demonstrated that E stimulates PRL synthesis and secretion in monkey pituitary cell cultures (8-10), suggesting that E can act directly on the primate lactotrope. Conversely, while P stimulates PRL secretion in the E-primed monkey (11), it has no effect on PRL secretion in E-treated pituitary cell cultures (9, 10). We Received August 15,1989. Address all correspondence and requests for reprints to: Dr. Cynthia L. Bethea, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. * Publication 1693 of the Oregon Regional Primate Research Center. This work was supported by NIH Grants HD-17269 (to C.L.B.) and HD-19182 (to R.M.B.), Reproductive Biology Training Grant HD07133 (to S.A.S.), P30 Program Project Grant HD-18185, and Grant RR-00163 for operation of the Oregon Regional Primate Research Center.
recently demonstrated that monkey lactotropes in culture lack progestin receptor (PR), which would account for the absence of a direct effect of P on PRL secretion (12). It is now important to extend this observation to the physiological environment of the whole animal. The effects of E and P on PRL secretion are expected to be mediated by their respective receptors. In the reproductive tract, E's actions on protein synthesis and growth are mediated by an increase in estrogen receptor (ER) synthesis and are accompanied by an induction of PR synthesis (13). Subsequent exposure to P decreases ER levels, and in this manner, P blocks the actions of E (14). P has also been reported to suppress its own receptor in the reproductive tract of E-primed monkeys (15). However, because P stimulates PRL secretion in the Eprimed monkey, it is also important to determine whether E and P modulate ER and PR levels in a similar fashion in the primate pituitary and reproductive tract. Therefore, the goals of the following series of studies are to 1) confirm the localization of PR in specific cell
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MONKEY PITUITARY ER AND PR
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types of the intact monkey pituitary with immunocytochemistry; 2) characterize the regulation of ER and PR by E and P in the nonhuman primate pituitary using biochemical assays incorporating monoclonal antibodies against ER and PR; and 3) relate receptor levels to steroid regulation of PRL secretion.
Materials and Methods Monkey selection and steroid treatment Thirteen regularly cycling adult cynomolgus macaque (Macaca fascicularis) females were exposed to a controlled steroid environment before measurement of ER and PR in the pituitary. At the time of ovariectomy (experimental day 0), each animal was placed into one of three steroid replacement groups by implanting steroid-filled Silastic capsules (16): 1) Spay, each animal (n = 5) received a 2-cm empty Silastic capsule sc; 2) E, each animal (n = 5) received a 2-cm E-filled capsule; and 3) E+P, each animal (n = 3) received a 2-cm E capsule at ovariectomy plus a 6-cm P-filled capsule on day 14. All animals were killed on experimental day 28. Daily blood samples (1.5 ml) were collected for E, P, and PRL determinations by RIA. The animals were housed and cared for according to procedures approved by the Animal Care and Use Committee of the Oregon Regional Primate Research Center (ORPRC). The anterior pituitary and endometrium were removed at autopsy for steroid receptor measurement and immunocytochemistry. Tissue treatment The pituitary was divided into three parts for ER measurement, PR measurement, and immunocytochemical (ICC) demonstration of cell type distribution of PR. The portion of pituitary tissue used for ICC was immediately frozen in liquid propane with O.C.T. (Miles Laboratories, Inc., Naperville, IL) and stored over liquid nitrogen until sectioned. The endometrium was dissected according to the method described by West et al. (16) and divided longitudinally for ER and PR measurements. ER and PR were measured in the fresh tissue with a gradient shift assay, which employs rat monoclonal antibodies to separate receptor-bound ligand from free and nonspecifically bound ligand on a 5-20% sucrose gradient (17,18). This receptor assay was chosen for its sensitivity and specificity. This assay has been used previously for the measurement of ER in monkey whole pituitary, pituitary cell cultures (10), and prostate (18). ER assay protocol For assay of ER, pituitary and endometrial tissue slices were incubated in Trowell's medium (Gibco, Grand Island, NY) with 10 nM [3H]E ([2,4,6,7,16,17-N-3H]E; New England Nuclear, Boston, MA) for 1 h at 37 C in an atmosphere of 60% O2-6% CO2-34% N2. After incubation, the slices were rinsed and homogenized in a low salt buffer [TEDGM; 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, 10% (vol/vol) glycerol, and 10 mM sodium molybdate, pH 7.4] using a Duall glass-glass tissue grinder. Homogenization and subsequent steps were carried out
Endo • 1990 Vol 126 • No 2
at 0-4 C. The homogenate was separated into crude nuclear and cytosolic fractions by centrifugation at 3,100 X g for 10 min. The pituitary cytosol was frozen for later measurement of PRL content by RIA (10). The nuclear pellets were washed three times with 1 ml TEDGM and finally resuspended in 500 til of a high salt buffer (TEDK; 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, and 0.5 M KC1, pH 7.4) and incubated for 1 h with frequent mixing to extract the steroid receptor from the chromatin. The extracted receptor and DNA were separated by centrifugation at 12,360 x g for 10 min. The pellets were frozen and later assayed for DNA by the method of Burton (19). The supernatant, containing the extracted receptor, was then incubated with 2.5 fig H222 (an ER-specific rat monoclonal immunoglobulin G (IgG) from Chris Nolan, Abbott Laboratories, Chicago, IL) for 1 h. The receptor-bound and free [3H]E were separated over a 5-20% sucrose gradient, prepared in TEK (TEDK without dithiothreitol), by ultracentrifugation for 18 h at 205,600 x g using a SW-41 rotor in a model L5-65 Beckman ultracentrifuge (Beckman Instrument Co., Palo Alto, CA). Twenty-five-drop fractions were collected after puncturing the bottom of the gradient tube. The radioactivity in each fraction was determined by adding 5 ml Scintiverse II (Fisher Scientific, Pittsburg, PA) and counting in a 460-CD Packard LSC (Packard, Downers Grove, IL). The [3H]ER-antibody complex separates cleanly from free and nonspecifically bound ligand (18). Receptor levels are calculated from the amount of radioactivity in the shifted peak using the specific activity of the [3H]E and the assumption of a 1:1 molar ratio between [3H]E and receptor. Receptor levels are reported as femtomoles of receptor per mg DNA. The sensitivity of the ER gradient shift procedure was estimated by assaying serial dilutions of the nuclear extract from the endometrium of an E-treated female monkey. A protein solution of BSA (0.1 mg/ml sedimenting at 4.5S) and aldolase (10 mg/ml sedimenting at 7.9S) was layered on a parallel gradient and used as a sedimentation control. Protein content in 25-drop fractions of the marker gradient was determined spectrophotometrically by measuring absorbance at 280 nm. Pi? assay validation and protocol A series of experiments using endometrial tissue were performed to validate and optimize the gradient shift assay for measurement of PR. Slices of endometrium and pituitary from an E-treated animal were labeled with 10 nM of the progestin analog [3H]R5020 ([17a-metfi;y/-3H]R5020; New England Nuclear, Boston, MA) under conditions identical to those used for ER except that the extracted receptor was incubated with 2.5 ng of the rat anti-PR monoclonal antibody B39 (Dr. Geoffery Greene, University of Chicago, Chicago, IL). In a second experiment, unincubated endometrial tissue was first homogenized in 700 /il TEDGM. Crude nuclear and cytosolic fractions were separated by centrifugation at 12,360 X g for 10 min. The cytosolic fraction was then incubated for 1 h with 1 nM [3H]R5020 at either 37 or 4 C. This was followed by incubation of 500 yul of the extract with 2.5 ng B39 for 1 h at 4 C. Ultracentrifugation and fraction collection were performed as described for ER.
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MONKEY PITUITARY ER AND PR In the final optimized assay, PR was measured in high salt extracts of unlabeled pituitary and endometrium to obtain total cellular PR. All steps were performed at 0-4 C to optimize antibody binding. Tissue was homogenized in 700 iA TEDK, and crude nuclear and cysolic fractions were obtained by centrifugation at 12,360 x g for 10 min. The extracted receptor in the cytosol fraction was labeled with 1 nM [3H]R5020 for 1 h, followed by a 1-h incubation with 2.5 jug B39. The nuclear pellets remaining after extraction were frozen for later DNA measurement (19). Layering of the complex over the sucrose gradients, centrifugation, fraction collection, and calculation of receptor content were identical to those described for the ER. The specificity of the assay was examined by the addition of a 100-fold excess of unlabeled R5020 or a 100-fold excess of unlabeled dexamethasone, or substitution for the anti-PR antibody with a rat monoclonal antibody (AT) of the same IgG subclass prepared against Timothy grass pollen (Dr. Arthur Malley, ORPRC). Assay sensitivity was estimated by assaying serial dilutions of a high salt extract from the endometrium of an E-treated female monkey. ICC procedure and analysis The frozen tissue blocks were mounted on microtome chucks with O.C.T., and sections were cut at 5 nm on a Hacker-Bright cryostat (Hacker Instruments, Inc., Fairfield, NJ) at -22 C. Tissue sections were mounted on slides coated with 0.1% polyL-lysine and transferred to precooled acetone for freeze substitution on the same day. The mounted sections remained in the acetone at —80 C for approximately 60 h. Preliminary experiments with pituitary tissue from random autopsy animals were performed to determine the optimal Cutting temperature. Pituitary ER and PR staining were improved by exposing the slides to the acetone freeze substitution Step immediately after sectioning and then processing through the ICC procedure. The amount of time in the acetone was not Critical, so exposure from 18-60 h was used. Pituitary tissue sections were fixed by a method similar to that described for pituitary cell cultures (12). After the acetone freeze-substitution, tissue sections were fixed at 4 C in picric acid-paraformaldehyde, followed by 85% ethanol; both contained 1.5% polyvinyl-pyrrolidone (PVP; mol wt, 360,000, Sigma Chemical Co., St. Louis, MO). Endogenous peroxidase activity in the tissue was blocked by the method of Andrew and Jasani (20). Sections were rinsed in 0.01 M PBS at room temperature, followed by a 45-min incubation in a blocking solution containing 1 mM sodium azide, 10 mM glucose, and 100 U glucose oxidase, and rinsed again in 0.01 M PBS at room temperature. After the blocking steps, sections were rinsed in 0.6 M PBS plus 1.5% PVP, incubated for 4 min in a 0.05% sodium borohydride solution plus 1.5% PVP, and rinsed in PBS containing 0.1% gelatin (PBS/gel) plus 1.5% PVP, all at 4 C. Identification of cells containing receptors and protein hormones was achieved by an indirect avidin-biotin-peroxidase method, using Vector ABC kits (Burlingame, CA). Tissue sections were incubated for 20 min at 4 C with nonimmune rabbit (for receptor staining) or goat serum (for protein staining). The slides were then incubated overnight at 4 C with the appropriate primary antibodies. ER was localized using a 10 Mg/ml mixture
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of the rat monoclonal antibodies H222 and D75 (Dr. Geoffrey Greene); the localization of PR was accomplished with 2.5 /ig/ ml B39 (Dr. Geoffrey Greene). The use of these antibodies for ER and PR immunocytochemistry has been previously documented (12, 21). Immunocytochemical localization of cellular PRL was accomplished with a 1:4000 dilution of a polyclonal rabbit antimonkey PRL antiserum (anti-MPRL-5) developed and characterized in this laboratory (12, 22). Cellular LH was identified with a 1:1000 dilution of a specific polyclonal rabbit antiserum obtained from NIH against human LHj3. The following day, the sections were washed with PBS/gel and again incubated with the nonimmune serum, followed by a 30-min incubation at room temperature with the appropriate biotinylated second antibodies. After additional washes in PBS/gel, each slide was incubated for 60 min at room temperature with the Vector ABC reagent. Antibody localization was detected by reaction with 35 mg/50 ml diaminobenzidine (Aidrich Chemical Co., Milwaukee, WI) plus 0.03% H2O2 for 5-20 min. The slides were rinsed and incubated for 1 min in 0.05% OsO4 to intensify the reaction product color development. The slides were again rinsed in water and postfixed for 3 min in picric acid-paraformaldehyde. Some slides were counterstained in hematoxylin in order to facilitate counting of negative cells. Tissue sections were then dehydrated in ethanol, infiltrated with xylenes, and coverslipped. To double stain for PR and one of the protein hormones, tissue sections were incubated with both primary antisera. The following day the slides were incubated with the secondary antibody against the protein hormone, followed by the secondary antibody against PR. The slides were then treated once with the ABC reagent and processed for color development in diaminobenzidine, followed by OsO4. Some variation in staining intensity was observed between different runs of the same tissue. Therefore, tissue sections from each of the three steroid treatment groups were processed for ER, PR, PRL, LH, PR+PRL, and PR+LH on the same day. This allowed for accurate comparisions of the effects of the steroid treatment on the percentage of positive cells and staining intensity. The percentage of positively stained cells was determined by counting positive and negative cells in four nonoverlapping fields in each tissue section (2-4 sections/animal) for a total of approximately 1000 cells/section. A mean percentage was then determined for each section and used in calculating mean percentages for each steroid treatment. PRL and steroid RIAs RIAs to measure serum E and P levels were performed by the P30 Radioimmunosaasy Laboratory core staff of ORPRC as previously described (23, 24) except for the source of the antibodies (25, 26). The monkey PRL RIA was performed in this laboratory as previously described (10, 22). Statistical treatments ER and PR levels and serum PRL, E, and P concentrations were compared by analysis of variance (ANOVA), followed by Duncan's multiple range test for significance of steroid treat-
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MONKEY PITUITARY ER AND PR
ment using the Clear Lake Research ANOVA package for the Macintosh PC (Houston, TX). Percentages of ER- or PR-positive cells from each steroid treatment were compared by ANOVA, followed by Duncan's multiple range test, to determine significance between treatments. Differences in staining intensity between steroid treatments on sections stained during the same ICC run were noted. Colocalization of PR with either PRL or LH was determined by observation.
Endo • 1990 Vol 126 • No 2
on serum PRL concentrations (overall average, 53.1 ± 4.6 ng/ml) compared to those in Spay animals (27.7 ± 2.3 ng/ml). However, the addition of P significantly (P < 0.05) increased PRL levels in the serum of E+P females (76.7 ± 7.3 ng/ml) compared to those in Spay animals. The pituitary content of PRL was not significantly affected by steroid treatment (Spay, 0.76 ± 0.23; E, 0.84 ± 0.26; E+P, 0.81 ± 0.23 Mg PRL/^g DNA). ER assay characteristics
Results Serum steroid levels Serum E levels were 9.5 ± 0.7 pg/ml in the Spay animals throughout the experiment. E levels averaged 310 ± 34.2 pg/ml in E- and E+P-treated monkeys immediately after capsule insertion and declined to 133.5 ± 5.0 pg/ml by day 5. Serum E was maintained in this range for the remainder of the experimental period. Serum P levels were 0.09 ± 0.01 ng/ml in all animals during the first 14 days of treatment. In E+P animals, serum P was elevated to 8.6 ± 0.4 ng/ml after insertion of the P capsule on day 14 and remained in this range for the duration of the experiment. Serum and pituitary PRL levels Serum PRL levels from the three groups over the 28 days of steroid treatment are shown in Fig. 1. Comparison of serum PRL levels during the final 14 days of treatment indicated that E alone had no significant effect
Figure 2 is an example of ER gradient profiles from the endometrium and pituitary of an E-treated animal. The [3H]ER-antibody complex separates cleanly from free and nonspecifically bound ligand, forming a peak of radioactivity in the 8S region of the gradient. The lowest detectable level of ER equaled 108 fmol/mg DNA in serial dilutions of endometrial nuclear extract from an E-treated animal. Characterization of the PR assay When endometrial and pituitary tissue slices from an E-treated animal were incubated at 37 C with [3H]R5020, and a nuclear extract was prepared, only background radioactivity was observed in the 8S region of the gradient (fractions 14-16), as shown in Fig. 3A. Therefore, this method of labeling and separating receptor was inappropriate for PR. When the whole cell extract was incubated with [3H] R5020 at 37 C, the receptor-specific peak of radioactivity
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DAY FIG. 1. Daily serum PRL levels in Spay, E-treated, and E+P-treated female monkeys. All animals were spayed on day 0 and received 2-cm empty (Spay) or E-filled capsules (E). Three of the E-treated monkeys (E+P) also received 6-cm P-filled capsules on day 14 (arrow). ANOVA, followed by Duncan's new multiple range test, indicated that E alone did not significantly increase PRL levels, but the addition of P significantly (P < 0.05) increased serum PRL levels above those observed in Spay animals. Bars indicating the SEM were omitted for clarity.
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FIG. 2. Example of an ER gradient shift assay of pituitary and endometrium from a spayed female monkey treated for 28 days with E. The shifted peak corresponds to the [3H]ER-antibody complex. BSA (4.5S; open arrow) and aldolase (7.9S; closed arrow) were run as markers on a parallel gradient in order to demonstrate the sedimentation characteristics of the gradients used in these experiments. Peak protein concentrations of the markers were detected by reading absorbance at 280 nm and are depicted by the arrows.
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MONKEY PITUITARY ER AND PR
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Nuclear Extract
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FIG. 4. Gradient profile of endometrial tissue homogenized in high salt buffer and labeled with [3H]R5020 at 4 C. Addition of a 100-fold excess of R5020, but not dexamethasone, abolished the shifted peak and increased the amount of radioactivity found in the unshifted region. Substitution of AT, an antibody of the same rat Ig subclass prepared against Timothy grass pollen, also blocked the formation of a shifted peak. These results verify the specificity of the ligand-receptor-antibody binding.
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FIG. 3. A, PR sucrose gradient profile prepared using endometrium and pituitary from an E-treated animal. Tissue slices were incubated with [3H]R5020 at 37 C for 1 h and homogenized in a low salt buffer, and PR was extracted from the nuclear fraction with a high salt buffer. No peak of radioactivity is observed in the 8S region, and only nonspecific binding was found above fraction 19. B, PR gradient shift assay of endometrial tissue homogenized in low salt buffer and then labeled with [3H]R5020 for 1 h at 37 or 4 C, followed by a 1-h incubation with 2.5 ng B39 at 4 C. A shifted peak of radioactivity was observed after incubation at 4 C but not 37 C. Closed arrows indicate 7.9S, and the open arrows indicate 4.5S, determined by aldolase and BSA sedimentation, respectively, as described in Fig. 2.
was still absent in the 8S region of the gradient. However, labeling of whole cell extract at 4 C resulted in the formation of a shifted peak of radioactivity and a clean Separation of the receptor-antibody complex from free and nonspecifically bound ligand (Fig. 3B). The presence of a 100-fold excess of unlabeled R5020 abolished the 8S peak and increased the amount of radioactivity found in the nonspecific peak (Fig. 4). This indicates that the binding in the shifted peak is progestin
specific, whereas the unshifted peak represents a high capacity binder with relatively low affinity. Substitution of the inappropriate rat monoclonal antibody, AT, for PR-specific B39 resulted in the absence of a shifted peak. Addition of a 100-fold excess of dexamethasone did not alter the gradient profile (Fig. 4); therefore, the glucocorticoid receptor is not competing for [3H]R5020 and contributing to the unshifted peak. The lowest detectable level of PR equaled 34.5 fmol/mg DNA in serial dilutions of a high salt extract from the endometrium of an Etreated monkey. Steroid regulation of ER and PR ER levels in the monkey pituitary were not significantly regulated by E or E+P (Fig. 5). Instead, pituitary ER were constitutively expressed independent of steroid influence. In the same animals endometrial ER were significantly affected by steroid treatment (P < 0.03, by ANOVA). ER levels were elevated by treatment with E for 28 days, but the range of values overlapped with that in the Spay group. However, the presence of P for 14 days significantly (P < 0.05) suppressed endometrial ER levels, as has been reported previously for this species (14). In contrast, pituitary PR levels were significantly (P < 0.03, by ANOVA) affected by steroid treatment (Fig. 6). Pituitary PR levels were undetectable (
Vol. 126, No. 2 Printed in U.S.A.
Regulation and Localization of Estrogen and Progestin Receptors in the Pituitary of Steroid-Treated Monkeys* S. A. SPRANGERS, N. B. WEST, R. M. BRENNER, AND C. L. BETHEA Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and the Departments of Physiology, and Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201
ABSTRACT. PRL increases during pregnancy in primates with rising levels of placental estradiol (E) and progesterone (P). However, while E will increase PRL secretion in monkey pituitary cell cultures, P has no effect. We recently localized progestin receptors (PR) to gonadotropes, but not lactotropes, with an immunocytochemical technique to double stain monkey pituitary cell cultures. The following studies were performed to confirm the immunocytochemical localization of PR in intact pituitary tissue and to determine the effect of E and P on the levels of estrogen receptors (ER) and PR in the pituitary. ER and PR levels were determined in the endometrium of the same animals for an internal comparison. Thirteen adult cycling female cynomolgus monkeys were ovariectomized and treated for 28 days with 1) an empty Silastic capsule (Spay), 2) a 2-cm Efilled capsule (E), or 3) a 2-cm E-filled capsule for 14 days plus a 6-cm P-filled capsule implanted for an additional 14 days (E+P). Blood samples were drawn daily for assay of serum E, P, and PRL levels. Serum PRL was not significantly affected by E, but the sequential addition of P significantly increased serum PRL levels over those observed in Spay animals. The anterior pituitary and endometrium were removed for measurement of
ER and PR levels by a sucrose gradient shift assay incorporating monoclonal antibodies against ER and PR. Pituitary ER levels did not vary significantly with steroid treatment (158.2 ± 33.6, 135.5 ± 24.9, 104.3 ± 13.4 fmol/mg DNA in Spay, E, and E+P animals, respectively). Pituitary PR levels were undetectable in Spay animals, were induced by E (393.3 ± 53.4 fmol/mg DNA), and were suppressed to undetectable levels by the addition of P. A portion of the pituitary was frozen for immunocytochemical single staining for ER, PR, PRL, and LH and double staining for PRL+PR and LH+PR. ER staining was observed in many parenchymal cells, but there was no apparent change with steroid treatment. PR staining was absent in the Spay animals; many PR-positive cells were observed in E-treated females, and only a small number of faintly staining cells were detected in the E+P animals. Double staining for PRL+PR and LH+PR revealed PR in gonadotropes, but not lactotropes. In conclusion, PR, but not ER, are regulated by E and P in the monkey pituitary. Importantly, PR is regulated within gonadotropes, but not lactotropes. Therefore, P probably increases PRL secretion through a hypothalamic action. (Endocrinology 126:1133-1142, 1990)
T N PRIMATES, serum PRL rises late in gestation JL coincident with rising estradiol (E) and progesterone (P) levels from placental sources (1, 2). E is a potent in uiuo stimulus for PRL secretion in rats (3-5), but E is less effective in stimulating PRL secretion in monkeys unless hypothalamic inhibition is first removed (6, 7). This laboratory has demonstrated that E stimulates PRL synthesis and secretion in monkey pituitary cell cultures (8-10), suggesting that E can act directly on the primate lactotrope. Conversely, while P stimulates PRL secretion in the E-primed monkey (11), it has no effect on PRL secretion in E-treated pituitary cell cultures (9, 10). We Received August 15,1989. Address all correspondence and requests for reprints to: Dr. Cynthia L. Bethea, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. * Publication 1693 of the Oregon Regional Primate Research Center. This work was supported by NIH Grants HD-17269 (to C.L.B.) and HD-19182 (to R.M.B.), Reproductive Biology Training Grant HD07133 (to S.A.S.), P30 Program Project Grant HD-18185, and Grant RR-00163 for operation of the Oregon Regional Primate Research Center.
recently demonstrated that monkey lactotropes in culture lack progestin receptor (PR), which would account for the absence of a direct effect of P on PRL secretion (12). It is now important to extend this observation to the physiological environment of the whole animal. The effects of E and P on PRL secretion are expected to be mediated by their respective receptors. In the reproductive tract, E's actions on protein synthesis and growth are mediated by an increase in estrogen receptor (ER) synthesis and are accompanied by an induction of PR synthesis (13). Subsequent exposure to P decreases ER levels, and in this manner, P blocks the actions of E (14). P has also been reported to suppress its own receptor in the reproductive tract of E-primed monkeys (15). However, because P stimulates PRL secretion in the Eprimed monkey, it is also important to determine whether E and P modulate ER and PR levels in a similar fashion in the primate pituitary and reproductive tract. Therefore, the goals of the following series of studies are to 1) confirm the localization of PR in specific cell
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MONKEY PITUITARY ER AND PR
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types of the intact monkey pituitary with immunocytochemistry; 2) characterize the regulation of ER and PR by E and P in the nonhuman primate pituitary using biochemical assays incorporating monoclonal antibodies against ER and PR; and 3) relate receptor levels to steroid regulation of PRL secretion.
Materials and Methods Monkey selection and steroid treatment Thirteen regularly cycling adult cynomolgus macaque (Macaca fascicularis) females were exposed to a controlled steroid environment before measurement of ER and PR in the pituitary. At the time of ovariectomy (experimental day 0), each animal was placed into one of three steroid replacement groups by implanting steroid-filled Silastic capsules (16): 1) Spay, each animal (n = 5) received a 2-cm empty Silastic capsule sc; 2) E, each animal (n = 5) received a 2-cm E-filled capsule; and 3) E+P, each animal (n = 3) received a 2-cm E capsule at ovariectomy plus a 6-cm P-filled capsule on day 14. All animals were killed on experimental day 28. Daily blood samples (1.5 ml) were collected for E, P, and PRL determinations by RIA. The animals were housed and cared for according to procedures approved by the Animal Care and Use Committee of the Oregon Regional Primate Research Center (ORPRC). The anterior pituitary and endometrium were removed at autopsy for steroid receptor measurement and immunocytochemistry. Tissue treatment The pituitary was divided into three parts for ER measurement, PR measurement, and immunocytochemical (ICC) demonstration of cell type distribution of PR. The portion of pituitary tissue used for ICC was immediately frozen in liquid propane with O.C.T. (Miles Laboratories, Inc., Naperville, IL) and stored over liquid nitrogen until sectioned. The endometrium was dissected according to the method described by West et al. (16) and divided longitudinally for ER and PR measurements. ER and PR were measured in the fresh tissue with a gradient shift assay, which employs rat monoclonal antibodies to separate receptor-bound ligand from free and nonspecifically bound ligand on a 5-20% sucrose gradient (17,18). This receptor assay was chosen for its sensitivity and specificity. This assay has been used previously for the measurement of ER in monkey whole pituitary, pituitary cell cultures (10), and prostate (18). ER assay protocol For assay of ER, pituitary and endometrial tissue slices were incubated in Trowell's medium (Gibco, Grand Island, NY) with 10 nM [3H]E ([2,4,6,7,16,17-N-3H]E; New England Nuclear, Boston, MA) for 1 h at 37 C in an atmosphere of 60% O2-6% CO2-34% N2. After incubation, the slices were rinsed and homogenized in a low salt buffer [TEDGM; 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, 10% (vol/vol) glycerol, and 10 mM sodium molybdate, pH 7.4] using a Duall glass-glass tissue grinder. Homogenization and subsequent steps were carried out
Endo • 1990 Vol 126 • No 2
at 0-4 C. The homogenate was separated into crude nuclear and cytosolic fractions by centrifugation at 3,100 X g for 10 min. The pituitary cytosol was frozen for later measurement of PRL content by RIA (10). The nuclear pellets were washed three times with 1 ml TEDGM and finally resuspended in 500 til of a high salt buffer (TEDK; 10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, and 0.5 M KC1, pH 7.4) and incubated for 1 h with frequent mixing to extract the steroid receptor from the chromatin. The extracted receptor and DNA were separated by centrifugation at 12,360 x g for 10 min. The pellets were frozen and later assayed for DNA by the method of Burton (19). The supernatant, containing the extracted receptor, was then incubated with 2.5 fig H222 (an ER-specific rat monoclonal immunoglobulin G (IgG) from Chris Nolan, Abbott Laboratories, Chicago, IL) for 1 h. The receptor-bound and free [3H]E were separated over a 5-20% sucrose gradient, prepared in TEK (TEDK without dithiothreitol), by ultracentrifugation for 18 h at 205,600 x g using a SW-41 rotor in a model L5-65 Beckman ultracentrifuge (Beckman Instrument Co., Palo Alto, CA). Twenty-five-drop fractions were collected after puncturing the bottom of the gradient tube. The radioactivity in each fraction was determined by adding 5 ml Scintiverse II (Fisher Scientific, Pittsburg, PA) and counting in a 460-CD Packard LSC (Packard, Downers Grove, IL). The [3H]ER-antibody complex separates cleanly from free and nonspecifically bound ligand (18). Receptor levels are calculated from the amount of radioactivity in the shifted peak using the specific activity of the [3H]E and the assumption of a 1:1 molar ratio between [3H]E and receptor. Receptor levels are reported as femtomoles of receptor per mg DNA. The sensitivity of the ER gradient shift procedure was estimated by assaying serial dilutions of the nuclear extract from the endometrium of an E-treated female monkey. A protein solution of BSA (0.1 mg/ml sedimenting at 4.5S) and aldolase (10 mg/ml sedimenting at 7.9S) was layered on a parallel gradient and used as a sedimentation control. Protein content in 25-drop fractions of the marker gradient was determined spectrophotometrically by measuring absorbance at 280 nm. Pi? assay validation and protocol A series of experiments using endometrial tissue were performed to validate and optimize the gradient shift assay for measurement of PR. Slices of endometrium and pituitary from an E-treated animal were labeled with 10 nM of the progestin analog [3H]R5020 ([17a-metfi;y/-3H]R5020; New England Nuclear, Boston, MA) under conditions identical to those used for ER except that the extracted receptor was incubated with 2.5 ng of the rat anti-PR monoclonal antibody B39 (Dr. Geoffery Greene, University of Chicago, Chicago, IL). In a second experiment, unincubated endometrial tissue was first homogenized in 700 /il TEDGM. Crude nuclear and cytosolic fractions were separated by centrifugation at 12,360 X g for 10 min. The cytosolic fraction was then incubated for 1 h with 1 nM [3H]R5020 at either 37 or 4 C. This was followed by incubation of 500 yul of the extract with 2.5 ng B39 for 1 h at 4 C. Ultracentrifugation and fraction collection were performed as described for ER.
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MONKEY PITUITARY ER AND PR In the final optimized assay, PR was measured in high salt extracts of unlabeled pituitary and endometrium to obtain total cellular PR. All steps were performed at 0-4 C to optimize antibody binding. Tissue was homogenized in 700 iA TEDK, and crude nuclear and cysolic fractions were obtained by centrifugation at 12,360 x g for 10 min. The extracted receptor in the cytosol fraction was labeled with 1 nM [3H]R5020 for 1 h, followed by a 1-h incubation with 2.5 jug B39. The nuclear pellets remaining after extraction were frozen for later DNA measurement (19). Layering of the complex over the sucrose gradients, centrifugation, fraction collection, and calculation of receptor content were identical to those described for the ER. The specificity of the assay was examined by the addition of a 100-fold excess of unlabeled R5020 or a 100-fold excess of unlabeled dexamethasone, or substitution for the anti-PR antibody with a rat monoclonal antibody (AT) of the same IgG subclass prepared against Timothy grass pollen (Dr. Arthur Malley, ORPRC). Assay sensitivity was estimated by assaying serial dilutions of a high salt extract from the endometrium of an E-treated female monkey. ICC procedure and analysis The frozen tissue blocks were mounted on microtome chucks with O.C.T., and sections were cut at 5 nm on a Hacker-Bright cryostat (Hacker Instruments, Inc., Fairfield, NJ) at -22 C. Tissue sections were mounted on slides coated with 0.1% polyL-lysine and transferred to precooled acetone for freeze substitution on the same day. The mounted sections remained in the acetone at —80 C for approximately 60 h. Preliminary experiments with pituitary tissue from random autopsy animals were performed to determine the optimal Cutting temperature. Pituitary ER and PR staining were improved by exposing the slides to the acetone freeze substitution Step immediately after sectioning and then processing through the ICC procedure. The amount of time in the acetone was not Critical, so exposure from 18-60 h was used. Pituitary tissue sections were fixed by a method similar to that described for pituitary cell cultures (12). After the acetone freeze-substitution, tissue sections were fixed at 4 C in picric acid-paraformaldehyde, followed by 85% ethanol; both contained 1.5% polyvinyl-pyrrolidone (PVP; mol wt, 360,000, Sigma Chemical Co., St. Louis, MO). Endogenous peroxidase activity in the tissue was blocked by the method of Andrew and Jasani (20). Sections were rinsed in 0.01 M PBS at room temperature, followed by a 45-min incubation in a blocking solution containing 1 mM sodium azide, 10 mM glucose, and 100 U glucose oxidase, and rinsed again in 0.01 M PBS at room temperature. After the blocking steps, sections were rinsed in 0.6 M PBS plus 1.5% PVP, incubated for 4 min in a 0.05% sodium borohydride solution plus 1.5% PVP, and rinsed in PBS containing 0.1% gelatin (PBS/gel) plus 1.5% PVP, all at 4 C. Identification of cells containing receptors and protein hormones was achieved by an indirect avidin-biotin-peroxidase method, using Vector ABC kits (Burlingame, CA). Tissue sections were incubated for 20 min at 4 C with nonimmune rabbit (for receptor staining) or goat serum (for protein staining). The slides were then incubated overnight at 4 C with the appropriate primary antibodies. ER was localized using a 10 Mg/ml mixture
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of the rat monoclonal antibodies H222 and D75 (Dr. Geoffrey Greene); the localization of PR was accomplished with 2.5 /ig/ ml B39 (Dr. Geoffrey Greene). The use of these antibodies for ER and PR immunocytochemistry has been previously documented (12, 21). Immunocytochemical localization of cellular PRL was accomplished with a 1:4000 dilution of a polyclonal rabbit antimonkey PRL antiserum (anti-MPRL-5) developed and characterized in this laboratory (12, 22). Cellular LH was identified with a 1:1000 dilution of a specific polyclonal rabbit antiserum obtained from NIH against human LHj3. The following day, the sections were washed with PBS/gel and again incubated with the nonimmune serum, followed by a 30-min incubation at room temperature with the appropriate biotinylated second antibodies. After additional washes in PBS/gel, each slide was incubated for 60 min at room temperature with the Vector ABC reagent. Antibody localization was detected by reaction with 35 mg/50 ml diaminobenzidine (Aidrich Chemical Co., Milwaukee, WI) plus 0.03% H2O2 for 5-20 min. The slides were rinsed and incubated for 1 min in 0.05% OsO4 to intensify the reaction product color development. The slides were again rinsed in water and postfixed for 3 min in picric acid-paraformaldehyde. Some slides were counterstained in hematoxylin in order to facilitate counting of negative cells. Tissue sections were then dehydrated in ethanol, infiltrated with xylenes, and coverslipped. To double stain for PR and one of the protein hormones, tissue sections were incubated with both primary antisera. The following day the slides were incubated with the secondary antibody against the protein hormone, followed by the secondary antibody against PR. The slides were then treated once with the ABC reagent and processed for color development in diaminobenzidine, followed by OsO4. Some variation in staining intensity was observed between different runs of the same tissue. Therefore, tissue sections from each of the three steroid treatment groups were processed for ER, PR, PRL, LH, PR+PRL, and PR+LH on the same day. This allowed for accurate comparisions of the effects of the steroid treatment on the percentage of positive cells and staining intensity. The percentage of positively stained cells was determined by counting positive and negative cells in four nonoverlapping fields in each tissue section (2-4 sections/animal) for a total of approximately 1000 cells/section. A mean percentage was then determined for each section and used in calculating mean percentages for each steroid treatment. PRL and steroid RIAs RIAs to measure serum E and P levels were performed by the P30 Radioimmunosaasy Laboratory core staff of ORPRC as previously described (23, 24) except for the source of the antibodies (25, 26). The monkey PRL RIA was performed in this laboratory as previously described (10, 22). Statistical treatments ER and PR levels and serum PRL, E, and P concentrations were compared by analysis of variance (ANOVA), followed by Duncan's multiple range test for significance of steroid treat-
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MONKEY PITUITARY ER AND PR
ment using the Clear Lake Research ANOVA package for the Macintosh PC (Houston, TX). Percentages of ER- or PR-positive cells from each steroid treatment were compared by ANOVA, followed by Duncan's multiple range test, to determine significance between treatments. Differences in staining intensity between steroid treatments on sections stained during the same ICC run were noted. Colocalization of PR with either PRL or LH was determined by observation.
Endo • 1990 Vol 126 • No 2
on serum PRL concentrations (overall average, 53.1 ± 4.6 ng/ml) compared to those in Spay animals (27.7 ± 2.3 ng/ml). However, the addition of P significantly (P < 0.05) increased PRL levels in the serum of E+P females (76.7 ± 7.3 ng/ml) compared to those in Spay animals. The pituitary content of PRL was not significantly affected by steroid treatment (Spay, 0.76 ± 0.23; E, 0.84 ± 0.26; E+P, 0.81 ± 0.23 Mg PRL/^g DNA). ER assay characteristics
Results Serum steroid levels Serum E levels were 9.5 ± 0.7 pg/ml in the Spay animals throughout the experiment. E levels averaged 310 ± 34.2 pg/ml in E- and E+P-treated monkeys immediately after capsule insertion and declined to 133.5 ± 5.0 pg/ml by day 5. Serum E was maintained in this range for the remainder of the experimental period. Serum P levels were 0.09 ± 0.01 ng/ml in all animals during the first 14 days of treatment. In E+P animals, serum P was elevated to 8.6 ± 0.4 ng/ml after insertion of the P capsule on day 14 and remained in this range for the duration of the experiment. Serum and pituitary PRL levels Serum PRL levels from the three groups over the 28 days of steroid treatment are shown in Fig. 1. Comparison of serum PRL levels during the final 14 days of treatment indicated that E alone had no significant effect
Figure 2 is an example of ER gradient profiles from the endometrium and pituitary of an E-treated animal. The [3H]ER-antibody complex separates cleanly from free and nonspecifically bound ligand, forming a peak of radioactivity in the 8S region of the gradient. The lowest detectable level of ER equaled 108 fmol/mg DNA in serial dilutions of endometrial nuclear extract from an E-treated animal. Characterization of the PR assay When endometrial and pituitary tissue slices from an E-treated animal were incubated at 37 C with [3H]R5020, and a nuclear extract was prepared, only background radioactivity was observed in the 8S region of the gradient (fractions 14-16), as shown in Fig. 3A. Therefore, this method of labeling and separating receptor was inappropriate for PR. When the whole cell extract was incubated with [3H] R5020 at 37 C, the receptor-specific peak of radioactivity
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DAY FIG. 1. Daily serum PRL levels in Spay, E-treated, and E+P-treated female monkeys. All animals were spayed on day 0 and received 2-cm empty (Spay) or E-filled capsules (E). Three of the E-treated monkeys (E+P) also received 6-cm P-filled capsules on day 14 (arrow). ANOVA, followed by Duncan's new multiple range test, indicated that E alone did not significantly increase PRL levels, but the addition of P significantly (P < 0.05) increased serum PRL levels above those observed in Spay animals. Bars indicating the SEM were omitted for clarity.
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FIG. 2. Example of an ER gradient shift assay of pituitary and endometrium from a spayed female monkey treated for 28 days with E. The shifted peak corresponds to the [3H]ER-antibody complex. BSA (4.5S; open arrow) and aldolase (7.9S; closed arrow) were run as markers on a parallel gradient in order to demonstrate the sedimentation characteristics of the gradients used in these experiments. Peak protein concentrations of the markers were detected by reading absorbance at 280 nm and are depicted by the arrows.
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MONKEY PITUITARY ER AND PR
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FIG. 4. Gradient profile of endometrial tissue homogenized in high salt buffer and labeled with [3H]R5020 at 4 C. Addition of a 100-fold excess of R5020, but not dexamethasone, abolished the shifted peak and increased the amount of radioactivity found in the unshifted region. Substitution of AT, an antibody of the same rat Ig subclass prepared against Timothy grass pollen, also blocked the formation of a shifted peak. These results verify the specificity of the ligand-receptor-antibody binding.
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FIG. 3. A, PR sucrose gradient profile prepared using endometrium and pituitary from an E-treated animal. Tissue slices were incubated with [3H]R5020 at 37 C for 1 h and homogenized in a low salt buffer, and PR was extracted from the nuclear fraction with a high salt buffer. No peak of radioactivity is observed in the 8S region, and only nonspecific binding was found above fraction 19. B, PR gradient shift assay of endometrial tissue homogenized in low salt buffer and then labeled with [3H]R5020 for 1 h at 37 or 4 C, followed by a 1-h incubation with 2.5 ng B39 at 4 C. A shifted peak of radioactivity was observed after incubation at 4 C but not 37 C. Closed arrows indicate 7.9S, and the open arrows indicate 4.5S, determined by aldolase and BSA sedimentation, respectively, as described in Fig. 2.
was still absent in the 8S region of the gradient. However, labeling of whole cell extract at 4 C resulted in the formation of a shifted peak of radioactivity and a clean Separation of the receptor-antibody complex from free and nonspecifically bound ligand (Fig. 3B). The presence of a 100-fold excess of unlabeled R5020 abolished the 8S peak and increased the amount of radioactivity found in the nonspecific peak (Fig. 4). This indicates that the binding in the shifted peak is progestin
specific, whereas the unshifted peak represents a high capacity binder with relatively low affinity. Substitution of the inappropriate rat monoclonal antibody, AT, for PR-specific B39 resulted in the absence of a shifted peak. Addition of a 100-fold excess of dexamethasone did not alter the gradient profile (Fig. 4); therefore, the glucocorticoid receptor is not competing for [3H]R5020 and contributing to the unshifted peak. The lowest detectable level of PR equaled 34.5 fmol/mg DNA in serial dilutions of a high salt extract from the endometrium of an Etreated monkey. Steroid regulation of ER and PR ER levels in the monkey pituitary were not significantly regulated by E or E+P (Fig. 5). Instead, pituitary ER were constitutively expressed independent of steroid influence. In the same animals endometrial ER were significantly affected by steroid treatment (P < 0.03, by ANOVA). ER levels were elevated by treatment with E for 28 days, but the range of values overlapped with that in the Spay group. However, the presence of P for 14 days significantly (P < 0.05) suppressed endometrial ER levels, as has been reported previously for this species (14). In contrast, pituitary PR levels were significantly (P < 0.03, by ANOVA) affected by steroid treatment (Fig. 6). Pituitary PR levels were undetectable (
