Role of Estrogen Receptor-{alpha} in the Anterior Pituitary Gland

Kathleen M. Scully, Anatoli S. Gleiberman, Jonathan Lindzey, Dennis B. Lubahn, Kenneth S. Korach and Michael G. Rosenfeld

Howard Hughes Medical Institute (K.M.S., A.S.G., M.G.R.), Department and School of Medicine, University of California, San Diego, La Jolla, California 92093-0648,
Receptor Biology Section (J.L., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709,
Departments of Biochemistry and Child Health (D.B.L.), University of Missouri, Columbia, Missouri 65211


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted insertional disruption of the mouse estrogen receptor-{alpha} (ER{alpha}) gene has provided a genetic model in which to test hypotheses that estrogens exert important effects in development and homeostatic functions of the anterior pituitary gland, particularly in the lactotroph and gonadotroph cell types. Analysis of ER{alpha} gene-disrupted mice reveals a marked reduction in PRL mRNA and a decrease in lactotroph cell number, but normal specification of lactotroph cell phenotype. Gonadotropin mRNA levels in ER{alpha} gene-disrupted female mice are elevated, consistent with previously described transcriptional suppression of gonadotropin subunit gene expression in response to sustained administration of estrogen in wild type mice. These results provide genetic evidence that ER{alpha} plays a critical role in PRL and gonadotropin gene transcription and is involved in lactotroph cell growth, but is not required for specification of lactotroph cell phenotype.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The five endocrine cell types of the mouse anterior pituitary gland — corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs — are derived during embryogenesis from a common primordium, known as Rathke’s pouch (1). Differentiation of these cell types proceeds in a distinct temporal and spatial pattern that is marked by cell type-specific transcriptional activation of genes encoding trophic hormones (2). Estrogen receptor (ER) has been detected as early as embryonic day 17 in the developing mouse pituitary (3), and, in the adult, relatively high levels of ER mRNA and protein are expressed in the lactotroph and gonadotroph cell types (4, 5).

In lactotroph cells, estrogens have been proposed to be involved in specification of cell phenotype, growth, and synthesis and secretion of PRL. Ontogenic analyses have correlated ER expression with the onset of PRL gene expression in the embryo and with a postnatal increase in lactotroph cell number (2, 6, 7, 8). Estrogens have been shown to stimulate lactotroph cell growth (9, 10) and PRL secretion (11, 12), as well as having been linked to the development of PRL-secreting pituitary tumors (13). A number of molecules have been hypothesized to mediate these effects: vasoactive intestinal peptide (14), galanin (15), and transforming growth factor-ß3 (16). Additionally, numerous lines of independent evidence indicate that mature lactotroph cells are derived from somatotroph cells, possibly through an intermediate cell type that coexpresses both GH and PRL (17, 18, 19, 20, 21, 22, 23, 24).

Ligand-bound ER has been demonstrated to activate PRL transcription via direct interaction with the PRL gene distal enhancer (25, 26, 27). Treatment with estrogen results in increased nuclease hypersensitivity of the PRL promoter and increased interaction of the promoter with the PRL distal enhancer (28, 29, 30). Transcriptional activation of PRL gene expression by ER involves synergism with the pituitary-specific POU domain factor, Pit-1, bound to a monomer DNA recognition site that dictates use of a tyrosine-dependent synergy domain in Pit-1 (2, 31, 32, 33).

Regulation of gonadotropin synthesis and secretion is mediated by GnRH from the hypothalamus, gonadal peptides, and classical feedback effects by gonadal steroids (34). Although an estrogen surge during proestrus can directly induce transcription of LHß mRNA (35), chronic treatment of animals with estrogens results in suppression of expression of all three gonadotropin subunit genes — LHß, FSHß, and {alpha}-glycoprotein subunit ({alpha}GSU) (36). The majority of evidence suggests that suppression of gonadotropin synthesis by chronic estrogen treatment is mediated indirectly via decreased hypothalamic GnRH secretion or altered pituitary responsiveness to GnRH (37, 38, 39, 40, 41, 42, 43, 44). However, the level at which estrogens regulate gonadotropin synthesis is species-dependent. In the rat, gonadotropin synthesis in isolated pituitaries in culture shows no response to estrogen while GnRH mRNA levels in cultured hypothalamic explants decrease in response to estrogen (34). In sheep, in contrast, estrogen has been shown to suppress FSHß subunit transcription in isolated pituitary cell cultures (45).

The ER{alpha} gene-disrupted mouse has provided a genetic model in which to test hypotheses regarding the role of estrogens in the development and function of the anterior pituitary (46). The ER{alpha} gene-disrupted mice undergo normal prenatal sexual development and survive to adulthood, but both genders are completely infertile. Mutant females possess hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea (46), have circulating estradiol levels that are 10-fold higher than wild type females (47), and exhibit alteration of gender-specific behaviors (48). Mutant males possess small testis, dysmorphogenic seminiferous tubules, and low sperm count (49). In this report, we examine the effect of a targeted insertional disruption of the mouse ER{alpha} gene on differentiation of endocrine cell types and trophic hormone gene expression in the anterior pituitary gland.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Trophic Hormone mRNA Levels in ER{alpha} Gene-Disrupted and Wild Type Female Mice
To assess the effect of ER{alpha} gene disruption on the expression of genes encoding markers of terminal differentiation, Northern analysis was used to compare steady state levels of mRNAs encoding all of the anterior pituitary trophic hormones in wild type and ER{alpha} gene-disrupted mice (Fig. 1Go). Total RNA was isolated from pooled pituitaries of wild type and ER{alpha} gene-disrupted 4-month-old non-parous female mice.



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Figure 1. Comparison of Trophic Hormone mRNA Levels in ER{alpha} Gene-Disrupted (ER-/-) and Wild Type (ER+/+) Female Mice

Northern analysis was used to compare total RNA from pituitaries of 4-month-old ER{alpha} gene-disrupted and wild type female mice. Total RNA (2.5 µg), approximately one pituitary equivalent, was loaded per lane for hybridization with all of the probes specific for the trophic hormone mRNAs except LHß (10 µg) and FSHß (20 µg). Each trophic hormone signal was normalized to the ß-actin signal, and the normalized values from the ER{alpha} gene-disrupted mice were divided by the normalized values from the wild type mice to give a ratio representing the change in mRNA levels as a result of ER{alpha} gene disruption.

 
The change in the level of PRL transcripts was the most dramatic, revealing a nearly 20-fold decrease in the ER{alpha} gene-disrupted mice relative to wild type controls, providing genetic evidence in support of a positive role for ER{alpha} in activation of PRL gene expression (25, 26, 27, 28, 29, 30, 31, 32, 33). Transcripts encoding all of the gonadotropin subunits were increased in the ER{alpha} gene-disrupted mice relative to wild type controls. Messenger RNA levels of the common {alpha}GSU subunit increased 4-fold in the ER{alpha} gene-disrupted mice, while transcripts encoding the unique subunits of the gonadotropins, LHß and FSHß, both increased almost 7-fold. This outcome is in agreement with data obtained in numerous studies using gonadectomy and estrogen replacement in which removal of the ovaries resulted in increased expression of all three gonadotropin subunit mRNAs, and subsequent administration of estrogen coordinately suppressed gonadotropin mRNA levels (50). Levels of POMC, TSHß, and GH mRNAs all increased to a small degree in the ER{alpha} gene-disrupted mice relative to wild type mice, although it is difficult to ascribe biological significance to these results.

Comparison of Gonadotropin and PRL mRNA Levels in ER{alpha} Gene-Disrupted and Ovariectomized Wild Type Mice to Intact Wild Type Mice
To evaluate the difference in the effect on anterior pituitary trophic hormone gene expression that resulted from hereditary ER{alpha} gene disruption vs. acute loss of estrogenic ligands, changes in steady state levels of mRNAs encoding the trophic hormones LHß, FSHß, and PRL from ER{alpha} gene-disrupted mice were compared by Northern analysis to the changes in mRNA levels that occurred in wild type mice of the same age and gender that had been ovariectomized 10 to 14 days before death (Fig. 2Go). In both the ER{alpha} gene-disrupted and the ovariectomized wild type mice, LHß and FSHß mRNAs increased to approximately equivalent levels compared to intact wild type mice. These data indicated that a transient decrease in estrogen produced by removal of the main site of estrogen synthesis, the ovaries, had essentially the same effect on the levels of LHß and FSHß mRNAs as hereditary ER{alpha} gene disruption. In the case of PRL transcripts, however, the decrease in steady state transcript level in the ovariectomized mice, approximately 2-fold, was not as large as the decrease seen in the ER{alpha} gene-disrupted mice, which was more than 10-fold. This result suggested that for PRL gene expression, the effect of hereditary ER{alpha} gene disruption was more profound than acute loss of estrogenic ligands.



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Figure 2. Comparison of Gonadotropin and PRL mRNA Levels in ER{alpha} Gene-Disrupted (ER-/-) and Ovariectomized Wild Type (OVEX) Mice to Intact Wild Type (ER+/+) Mice

Northern analysis was used to compare total RNA from pituitaries of 4-month-old female ER{alpha} gene-disrupted and ovariectomized wild type mice to intact wild type control mice. Ten micrograms of total RNA were loaded for analysis with LHß and PRL probes, and 20 µg were loaded for analysis with the FSHß probe. Each trophic hormone signal was normalized to the ß-actin signal, and the normalized values from the ER{alpha} gene-disrupted and ovariectomized wild type mice were divided by the normalized values from the intact wild type mice to give a ratio representing the change in mRNA levels as a result of ER{alpha} gene disruption or ovariectomy.

 
PRL mRNA Levels in ER{alpha} Gene-Disrupted and Wild Type Male Mice
The first case of a mutation in the human ER, a single base pair change in the second exon that generated a premature stop codon, was recently reported in a 28-yr-old male (51). Biochemical measurement of serum levels of anterior pituitary hormones in this patient revealed significantly increased levels of LH and FSH, but PRL serum concentrations in the normal range. Although posttranscriptional regulation might have compensated for a decrease in PRL mRNA in the human male, it was of interest to examine male ER{alpha} gene-disrupted mice to identify a possible gender difference in the requirement for ER{alpha} in generation of normal PRL mRNA levels. Northern analysis was used to compare the steady state level of PRL mRNA in ER{alpha} gene-disrupted mice and wild type control mice (Fig. 3Go). As in the case of the female ER{alpha} gene-disrupted mice, however, there was a large decrease of approximately 10-fold in the PRL mRNA levels of male ER{alpha} gene-disrupted mice.



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Figure 3. Comparison of PRL mRNA Levels in ER{alpha} Gene-Disrupted (ER-/-) and Wild Type (ER+/+) Male Mice

Northern analysis was used to compare total RNA from pituitaries of 4-month-old male ER{alpha} gene-disrupted to wild type mice. Five micrograms of total RNA were loaded per lane. Each PRL signal was normalized to the ß-actin signal, and the normalized values from the ER{alpha} gene-disrupted mice were divided by the normalized values from the wild type mice to give a ratio representing the change in PRL mRNA level as a result of ER {alpha} gene disruption in male mice.

 
Immunohistochemical Comparison of Trophic Hormones in the Anterior Pituitaries of Wild Type and ER{alpha} Gene-Disrupted Mice
To assess the effect of ER{alpha} gene disruption on growth of specific cell types and secretion of trophic hormones, pituitary glands of wild type and ER{alpha} gene-disrupted adult female mice were sectioned and immunostained with antisera directed against the trophic hormones elaborated by each of the five endocrine cell types. The patterns of immunostaining of all three gonadotropin subunit proteins ({alpha}GSU, LHß, and FSHß) were altered in the ER{alpha} gene-disrupted mice relative to wild type controls (Fig. 4AGo). Immunostaining of {alpha}GSU, the common subunit of LH, FSH, and TSH, revealed a moderate increase in cell density in the ER{alpha} gene-disrupted mice relative to wild type control mice. An increase in cell density was also noted in the ER{alpha} gene-disrupted mice with TSHß immunostaining, but no change was observed with LHß or FSHß immunostaining. In the case of LHß, the intensity of cytoplasmic staining in the ER{alpha} gene-disrupted mice was significantly decreased relative to wild type mice, suggesting that ER{alpha} may normally exert a suppressive effect on LH secretion. FSHß immunostaining in the ER{alpha} gene-disrupted mice was similar to the wild type mice with the exception of a small number of cells in the ER{alpha} gene-disrupted mice that displayed increased intensity of cytoplasmic staining.



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Figure 4. Immunohistochemical Staining of Trophic Hormones in ER{alpha} Gene-Disrupted (ER-/-) and Wild-Type (ER+/+) Mice

Coronal sections from adult female ER{alpha} gene-disrupted (n = 3) and wild type (n = 3) mice were immunostained with primary antisera directed against pituitary trophic hormones. Secondary antibodies were coupled to horseradish peroxidase. Sections were reacted with peroxide and diaminobenzidine as the chromogen and counter stained with methyl green. A, Gonadotropin subunit immunostaining. B, ACTH, TSHß, GH, and PRL immunostaining.

 
PRL immunostaining revealed a modest decrease in lactotroph cell density, suggesting that, in agreement with existing data, ER{alpha} normally exerts a positive effect on lactotroph cell growth (Fig. 4BGo). Among immunoreactive PRL cells, however, no difference in intensity of cytoplasmic staining was observed between ER{alpha} gene-disrupted and wild type mice. Immunostaining with antisera directed against ACTH revealed a moderate increase in cell density in ER{alpha} gene-disrupted mice relative to wild type controls, but no change was detected with GH antisera.

Lactotroph Cell Phenotype Specification in ER{alpha} Gene-Disrupted Mice
Although it has been hypothesized that lactotrophs are derived from somatotrophs, possibly through an intermediate somatolactotroph cell type that coexpresses GH and PRL (17, 18, 19, 20, 21, 22, 23, 24), transcription factors involved in switching trophic hormone gene expression from GH to PRL have not been conclusively identified. ER is a candidate factor for such a role because its ontogeny parallels the ontogeny of PRL expression (6, 7, 8), and because ER positively regulates PRL transcription (25, 26, 27, 28, 29, 30, 31, 32, 33). If ER{alpha} functioned in such a capacity, mice harboring an ER{alpha} gene disruption might lack mature lactotrophs that express only PRL and not GH. To assess the effect of ER{alpha} gene disruption on progression of the somatotroph-lactotroph lineage, pituitary sections from adult ER{alpha} gene-disrupted female mice were simultaneously immunostained with antisera directed against both GH and PRL. Results were visualized using dual indirect immunofluorescence (Fig. 5Go). Specification of lactotroph cell phenotype appeared to be unaffected in the ER{alpha} gene-disrupted mice based on the readily observable immunostaining of numerous cells that express PRL but do not coexpress GH.



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Figure 5. Immunohistochemical Analysis of the Somatotroph-Lactotroph Cell Lineage in ER{alpha} Gene-Disrupted (ER-/-) Mice

Double labeling of coronal sections from adult female ER{alpha} gene-disrupted (ER-/-) mice (n = 3) with primary antisera directed against GH and PRL. Secondary antibody directed against the GH antisera was labeled with rhodamine (red) and against the PRL antisera with fluorescein (green). Arrowheads indicate cells that labeled with PRL but not GH antisera.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of trophic hormone gene expression in ER{alpha} gene-disrupted mice provides genetic evidence for the long-held hypothesis that a major effect of estrogen in the anterior pituitary gland is to increase PRL gene expression (25, 26, 27, 28, 29, 30, 31, 32, 33). This may result from a direct effect on PRL gene transcription and an effect on lactotroph cell growth. Comparison of PRL mRNA levels in ER{alpha} gene-disrupted and ovariectomized wild type mice to intact wild type mice revealed a greater decrease in PRL mRNA level in the ER{alpha} gene-disrupted mice than in ovariectomized wild type mice, suggesting that ER{alpha} gene disruption results in a more profound effect on PRL gene expression than acute loss of estrogenic ligands. In support of a role for estrogens in lactotroph cell growth (6, 7, 8, 9, 10), immunohistochemical staining of the pituitary glands of ER{alpha} gene-disrupted mice with PRL antisera revealed a modest decrease in lactotroph cell density. Despite the decrease in PRL mRNA, there was no discernable decrease in cytoplasmic PRL immunoreactivity in individual lactotroph cells in sections from ER{alpha} gene-disrupted mice, in support of a role for ER{alpha} in estrogen-stimulated secretion of PRL. The overall decrease in the size of the anterior pituitary gland noted in the ER{alpha} gene-disrupted mice may result from decreased lactotroph cell number and may, in turn, explain the apparent relative increase in density of the ACTH-, {alpha}GSU-, and TSHß-positive cells.

Another potential role for estrogens in the anterior pituitary was specification of lactotroph cell phenotype (8). Several lines of independent evidence indicate that the GH-producing somatotrophs and the PRL-producing lactotrophs are lineally related, with somatotrophs giving rise to mature lactotrophs (17, 18, 19, 20, 21, 22, 23, 24, 52). Additionally, because of the parallel ontogeny of ER and PRL gene expression (2), and the positive effect of estrogen on PRL gene transcription (25, 26, 27, 28, 29, 30, 31, 32, 33) and lactotroph cell growth (6, 7, 8, 9, 10), it was of interest to examine the effect of ER{alpha} gene disruption on progression of the somatotroph-lactotroph lineage. Dual indirect immunofluorescence experiments with antisera directed against GH and PRL revealed a large number of cells staining for PRL, but not GH, in the ER{alpha} gene-disrupted mice, indicating that ER{alpha} is not required for specification of lactotroph cell phenotype as the somatotroph-lactotroph lineage progresses.

Increased gonadotropin hormone mRNA levels in ER{alpha} gene-disrupted mice have provided support for previous findings indicating that estrogens suppress synthesis of {alpha}GSU, LHß, and FSHß subunit mRNAs (36, 37, 38, 39, 40, 41, 42, 43, 44). Furthermore, when compared to wild type mice, an increase of similar magnitude in LHß and FSHß mRNA levels was observed in ER{alpha} gene-disrupted and ovariectomized wild type mice, suggesting that ER{alpha} gene disruption produced the same result as acute loss of estrogenic ligands. Immunohistochemical staining of the anterior pituitaries of ER{alpha} gene-disrupted mice with LHß and FSHß antisera revealed no change in gonadotroph cell density, but did show a notable decrease in LHß cytoplasmic immunoreactivity, compatible with a possible posttranscriptional role for ER{alpha}.

ER{alpha} gene-disrupted mice are a valuable model for the study of estrogen insensitivity in the anterior pituitary gland, as demonstrated by the profound effects on PRL and gonadotropin gene expression documented in the ER{alpha} gene-disrupted mice. The more modest effect on lactotroph cell growth observed in the ER{alpha} gene-disrupted mice could reflect limited biological dependence of lactotroph cell growth on ER{alpha}, expression of variant ER{alpha} transcripts in the ER{alpha} gene-disrupted mice, or a possible role for the recently discovered novel ER, ERß (53, 54, 55). ERß mRNA was not detected, however, by RNase protection assay of pituitaries from either wild type or ER{alpha} gene-disrupted mice, diminishing the likelihood that ERß mediates estrogen effects in the pituitary (J. Couse and K. Korach, unpublished data). RNase protection assays with a probe to the ligand-binding domain of ER{alpha}, in contrast, detected 10–20% of wild type levels of ER{alpha} mRNA in pituitaries from ER{alpha} gene-disrupted mice (J. Couse and K. Korach, unpublished data). It has not been proven whether this ER{alpha} mRNA represents a variant transcript, such as the E1 transcript identified in the uterus of ER{alpha} gene-disrupted mice (47), or the estrogen-inducible female- and tissue-specific transcripts reported in the pituitary of the rat (56). In the uterus of the ER{alpha} gene-disrupted mice, however, despite the presence of low levels of the E1 variant transcript, there was no increase of known uterine markers of estrogen action in response to treatment with estrogen, indicating that the presence of the E1 transcript did not result in any biologically significant response to estrogen in this organ (47). In the pituitary, given the pronounced effect of ER{alpha} gene disruption on PRL and gonadotropin gene expression, it seems likely that the more modest effect on lactotroph cell density may reflect a limited requirement for ER{alpha} in normal lactotroph cell growth.

In summary, these genetic data argue that the major effects of ER{alpha} in the anterior pituitary gland are in regulation of PRL and gonadotropin gene expression. The data also suggest a limited role for ER{alpha} in lactotroph cell growth and possible involvement of ER{alpha} in PRL and LH secretion, but no requirement for ER{alpha} in specification of the lactotroph cell phenotype.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Northern Blot Analysis
Total RNA was isolated by the acid-phenol method from pituitaries of 4-month-old female (male, Fig. 3Go) mice. Pituitaries were excised and frozen on dry ice, then lysed in Solution D (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol) in a volume of 0.5 ml per pituitary. Sodium acetate, pH 4.0, was added to 0.2 M final concentration, and the mixture was extracted with an equal volume of water-saturated phenol and 0.2 volume of chloroform. The aqueous phase was precipitated with an equal volume of isopropanol, resuspended in 15% of the original volume of Solution D, and precipitated again with an equal volume of isopropanol. The precipitate was washed twice with 75% ethanol and resuspended in water. The RNA was size fractionated on 1.1% formaldehyde/1.2% agarose gels, transferred to nylon membranes, and hybridized overnight at 65 C to DNA probes random-primed-labeled with Klenow fragment and 32P to a specific activity of 109 cpm/µg. The membranes were washed in 0.3XxNaCl-sodium citrate (SSC) for 45 min at 65 C and exposed to x-ray film. After x-ray film development, the blots were stripped and rehybridized with a 32P-labeled ß-actin DNA probe. For quantification, phosphor screens were exposed to the blots, and the imaged signals for trophic hormone mRNAs were normalized to the ß-actin signal.

Immunohistochemical Analysis
Mice were perfused transcardially with 20 ml PBS followed by 30 ml 10% formalin. Pituitaries were excised, postfixed in ethanol-37% formaldehyde-H20 water (6:1:3) for 1 h at room temperature, washed three times in 70% ethanol, and stored in 70% ethanol at 4 C. Pituitaries were dehydrated in isopropanol and toluene and embedded in paraffin. Five-micrometer thick sections were cut, mounted, and rehydrated in toluene and ethanol. All further steps were carried out in a solution of PBS and 0.05% TritonX-100. Sections were blocked with 10% normal goat serum and immunostained with polyclonal antisera for 1 h at room temperature. Antisera from the National Hormone and Pituitary Program (NIDDK, Rockville, MD) were directed against rat TSHß (AFP-1274789) used at a dilution of 1:10,000, rat LHß (AFP-2223879OGPOLHB) used at 1:12,500, and human FSHß (AFP-891891) used at 1:250. Antiserum against human ACTH (Sigma Chemical Co., St. Louis, MO) was used at 1:1000. Antisera against human GH and human PRL (DAKO, Santa Barbara, CA) were used at 1:200. Secondary antibodies coupled to horseradish peroxidase and directed against guinea pig (DAKO) or rabbit (Amersham, Arlington Heights, IL) were used at 1:100. Diaminobenzidine was the chromogen supplied as a diaminobenzidine/metal concentrate mixed with stable peroxidase substrate buffer (Pierce, Rockford, IL). Sections were counterstained with methyl green. All comparative immunostaining data are derived from ER{alpha} gene-disrupted and wild type mouse pituitary sections mounted on the same slides to ensure uniformity of processing.

Antisera directed against rat GH from the National Hormone and Pituitary Program (AFP 411S) was used at a dilution of 1:3000 and detected with secondary antibody coupled to rhodamine (Cappel, Durham, NC) used at 1:200. Antisera directed against human PRL (Vector, Burlingame, CA) was used at a dilution of 1:250 and detected with secondary antibody coupled to fluorescein (American Qualex, San Clemente, CA) used at 1:100.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Aimee Ryan, Dr. Bogi Andersen, and Daniel Szeto for critical reading and constructive comments during preparation of this manuscript. We also thank the National Hormone and Pituitary Program, NIDDK (Rockville, MD), and Dr. A. F. Parlow for generously providing antisera to anterior pituitary trophic hormones.


    FOOTNOTES
 
Address requests for reprints to: Michael G. Rosenfeld, University of California, San Diego, CMM-West Room 353, 9500 Gilman Drive, La Jolla, California 92093-0648.

Received for publication February 26, 1997. Accepted for publication March 24, 1997.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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