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Endocrinology Vol. 144, No. 10 4626-4636
Copyright © 2003 by The Endocrine Society

Targeted Overexpression of Luteinizing Hormone Causes Ovary-Dependent Functional Adenomas Restricted to Cells of the Pit-1 Lineage

Helai P. Mohammad, Rula A. Abbud1, Al F. Parlow, Jonathan S. Lewin and John H. Nilson

Department of Pharmacology, Case Western Reserve University (H.P.M., R.A.A., J.H.N.), Cleveland, Ohio 44106; National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center (A.F.P.), Torrance, California 90509; and Department of Radiology and Oncology, University Hospitals of Cleveland and Case Western Reserve University (J.S.L.), Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: John H. Nilson, Ph.D., Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106. E-mail: jhn{at}cwru.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The majority of pituitary adenomas in humans are nonmetastasizing, monoclonal neoplasms that occur in approximately 20% of the general population. Their development has been linked to a combination of extrinsic factors and intrinsic defects. We now demonstrate with transgenic mice that targeted and chronic overexpression of LH causes ovarian hyperstimulation and subsequent hyperproliferation of Pit-1-positive cells that culminates in the appearance of functional pituitary adenomas ranging from focal to multifocal expansion of lactotropes, somatotropes, and thyrotropes. Tumors fail to develop in ovariectomized mice, indicating that contributions from the ovary are necessary for adenoma development. Although the link between chronic ovarian hyperstimulation and PRL-secreting adenomas was expected, the involvement of somatotropes and thyrotropes was surprising and suggests that multiple ovarian hormones may contribute to this unusual pathological consequence. In support of this idea, we have found that ovariectomy followed by estrogen replacement results in the expansion of lactotropes selectively in LH overexpressing mice, but not somatotropes and thyrotropes. Collectively, these data indicate that estrogen is sufficient for the formation of lactotrope adenomas only in animals with a hyperstimulated ovary, whereas the appearance of GH- and TSH-secreting adenomas depends on multiple ovarian hormones. Together, our data expand current models of pituitary tumorigenesis by suggesting that chronic ovarian hyperstimulation may underlie the formation of a subset of pituitary adenomas containing lactotropes, somatotropes, and thyrotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY TUMORS ARE commonly occurring neoplasms present in approximately 20% of the general population, with a higher frequency in females. Despite their usual classification as benign, pituitary adenomas can be difficult to diagnose and manage, and they cause both morbidity and mortality in patients (1, 2, 3). In addition, the pathogenesis underlying the formation of pituitary adenomas remains controversial. Two prevailing theories on the mechanisms that underlie pituitary adenoma development argue for either an intrinsic lesion, such as a molecular defect, or an extrinsic influence, such as hormonal stimulation or growth factor support. Ultimately, the two current theories can be integrated by invoking a multistep process that leads to transformation resulting from both extrinsic cues and intrinsic events (2, 3).

In recent years several candidate protooncogenes and tumor suppressors have been linked to the pathogenesis of pituitary tumors through the use of transgenic and nude mouse models and analyses of human tumor samples (reviewed in Refs. 2, 3 , and 5). Several mouse models, including mice deficient in p27 (6, 7), Rb (8), dopamine receptors (D2R) (9), and PRL (10, 11) as well as those overexpressing genes such as TGF{alpha} (12) or GHRH (13, 14), have developed pituitary tumors. Overexpression of protooncogenes, such as PTTG, in immortalized cell lines has also been shown to induce transformation and results in tumor growth when these cells are injected into nude mice (15). However, many of the pathways implicated in either mouse or in vitro model systems do not correlate with human tumors. As a consequence, the current tumor markers still fail to adequately explain the mechanisms by which most pituitary tumors develop in humans (3). This underscores the need for further characterization of the molecular events surrounding the transformation of pituitary cells as well as the mechanisms that regulate the growth and proliferation of cells in the pituitary gland.

Clinical reports often suggest that human pituitary adenomas are rarely accompanied by hyperplasia of the adenohypophysis (2), supporting the idea of an intrinsic lesion as the cause of adenoma. However, pituitary hyperplasia has been documented in patients with Carney complex somatotropinomas as well as in tumors with deletions in multiple endocrine neoplasm-1 (16, 17). Horvath and colleagues (18) reported adenoma development with hyperplasia of somatotropes, lactotropes, corticotropes, and thyrotropes. Thus, it is plausible that hyperplasia may accompany or precede at least some pituitary adenomas in humans; this points to the potential importance of extrinsic factors.

Extrinsic factors that underlie hyperplasia are not well understood. It is believed, however, that hypothalamic releasing hormones that both stimulate hormone release and have mitogenic effects (e.g. CRH and GHRH) can provide sufficient external stimuli for increases in cell number (14, 19, 20, 21). Additionally, the loss of negative hormonal feedback, as in the case of hypothyroidism, has been implicated in pituitary hyperplasia in humans (18). Estrogen has also been implicated as an extrinsic factor that underlies the development of pituitary tumors in both humans and rodents. For example, it is clear that the estrogen receptor has an important physiological role in the regulation of PRL biosynthesis and lactotrope cell number, because mice lacking estrogen receptor {alpha} ({alpha} ERKO) have a reduction in both (22). In addition, estrogen treatment has been shown to result in the proliferation of lactotropes both in vivo and in vitro (23, 24, 25, 26, 27, 28, 29), and increased levels of estrogen during pregnancy have been correlated with expansion of the pituitary gland (26, 27, 28). Moreover, pharmacological doses of estrogen have been shown to cause prolactinomas in Fischer 344 rats (30, 31). Together, these data suggest that extrinsic factors may be mitogenic as well as play a role in pituitary adenoma formation.

To further evaluate the role of extrinsic factors in development of pituitary adenomas, we generated a transgenic mouse that hypersecretes LH (LHCTP) and develops numerous subsequent pathologies (32, 33), including multicystic ovaries, strain-dependent granulosa cell tumors (34, 35, 36), mammary cancer (37), and elevated steroid hormones, including estrogen (38, 39, 40). Elevated LH has been associated with polycystic ovarian syndrome and ovarian tumors in women (41, 42). However, a potential link between elevated serum LH and pituitary pathology remains largely unexplored. Herein we demonstrate that ovarian hyperstimulation induced by chronically elevated LH leads to hyperproliferation of Pit-1-positive cells, followed by subsequent adenoma development restricted to somatotropes, thyrotropes, and lactotropes. Our studies establish a unique model of pituitary hyperplasia and ovarian-dependent adenomas restricted to cells of the Pit-1 lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic mice were generated by breeding male CF-1 transgenics (LHCTP) to wild-type female mice and were genotyped as previously described (34). The males were at least 10 generations from the founder animal. All mice used in these studies were the F1 progeny of male CF-1 transgenics (LHCTP) and wild-type FVB/N females. Animals were maintained in the Animal Resource Center and were housed in microisolator units with a 12-h light, 12-h dark schedule and constant temperature. Surgeries, including ovariectomy and retroorbital blood collection, were performed under avertin anesthesia. Animals were anesthetized with avertin [tribromoethanol:tert-amylalcohol stock (1:1) diluted to 2.5% in 0.9% saline] for all survival procedures. Tissue samples and cardiac blood were obtained after mice were killed with CO2 gas. All studies involving animals were approved by the CWRU institutional animal care and use committee.

Ovariectomy/estradiol replacement
Transgenic and wild-type female mice were subjected to ovariectomy or sham surgery at two time points. Mice that underwent surgery at 6 wk of age were killed at 20 wk of age, and mice that underwent surgery at 16 wk were killed at 10 months of age. During surgery, 16-wk-old mice were implanted with either constant release pellets containing 17ß-estradiol (0.05 mg/pellet, 90-d release) or placebo (Innovative Research of America, Sarasota, FL). This dosage was determined before the study in a dose-response analysis (data not shown). Pellets were replaced at both 24 and 32 wk to maintain elevated estradiol levels. Retroorbital blood was collected at the time of surgery and pellet implantation for the measurement of serum estradiol.

Tissue collection
Pituitaries were removed immediately after death, weighed, and fixed in 4% paraformaldehyde for 2–4 h. The tissue was then paraffin-embedded and sectioned at 5 µm. Staining with hematoxylin and eosin was performed on some sections.

Reticulum staining
Gordon-Sweet silver staining was performed using reticulum staining reagents (Sigma-Aldrich Corp., St. Louis, MO) with methyl-green counterstain. A total of seven tumors from transgenic females and four age-matched wild-type pituitaries were used in two separate experiments.

Immunohistochemistry
Pituitary sections were deparaffinized in Xylene (Sigma-Aldrich Corp.) or Citrus Clearing Solvent (Richard Allan Scientific, Kalamazoo, MI) and rehydrated in 100% and 95% ethanol, followed by water. Antigen retrieval in 0.01 M citrate buffer was performed for immunostaining with anti-Pit-1 and antiproliferating cell nuclear antigen (anti-PCNA) antibodies. All sections were incubated in a humidified chamber for 60 min at room temperature in a blocking solution containing 1x PBS, BSA (0.05 g/ml), and normal goat serum (15 µl/ml), corresponding to the species in which the secondary antibody was raised. Sections were then incubated with the following primary antibodies: guinea pig antirat LH (National Hormone and Pituitary Program; 1:200), monkey antirat GH (National Hormone and Pituitary Program; 1:200), rabbit antirat PRL (National Hormone and Pituitary Program; 1:200), rabbit antihuman ACTH (National Hormone and Pituitary Program; 1:200), rabbit antimouse TSH (National Hormone and Pituitary Program; 1:1000), rabbit antihuman Pit-1 (gift from Simon Rhodes; 1:500), and mouse antihuman PCNA (PC10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:500), diluted in PBS/BSA. Primary antibody incubation took place at 37 C for 1–3 h. Sections were then incubated with the following secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated goat antirabbit (1:300), Alexa 594-conjugated goat antimouse (1:500), FITC-conjugated goat antiguinea pig (1:300), and FITC-conjugated goat antimonkey (1:300) at 37 C for 1 h. For all secondary antibodies used, a no-primary antibody control was performed to ensure the specificity of the signal. Antibodies used in double labeling were applied concomitantly. Sections were then washed in 0.05% PBS/Tween 20 for 20 min at room temperature after each antibody incubation. 4',6-Diamido-2-phenylindole hydrochloride and propidium iodide stains (when used) were present in the aqueous mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA). Images were collected, and Metamorph (Universal Imaging Corp., Downingtown, PA) was used to generate standard area counts for stained nuclei. Hormone-positive cells were counted manually and normalized to the total number of cells per field, as counted by Metamorph. At least two representative images were collected at x40 for each stain from each animal and were used in quantitative analyses. Due to the concentrated nature of the TSH-positive cells, for TSH staining, all positive cells in the entire section (at least two sections per animal) were counted and normalized to the total number of nuclei per field. Similarly, for PCNA staining, all positive cells in the entire section (at least two sections per animal) were counted and normalized to the total number of Pit-1-positive cells per field, because every PCNA-positive cell was also Pit-1 positive. All cell counts were performed blind.

RIAs
Steroid hormone RIAs were performed using assays from Pantex (Santa Monica, CA) that were previously validated in our laboratory for use with mouse serum (43, 44). The limit of detection for serum levels of estradiol for this assay is 10 pg/ml. PRL, LH, GH, and TSH levels were measured by RIA on serum samples taken from the same animal. Sera were shipped on dry ice to Dr. Al Parlow at National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center, who performed all RIAs. The antibodies used for PRL, GH, LH, and TSH assays were antimouse PRL AFP131078 (1:500,000), NIDDK antirat GH-RIA-5(AFP) (1:3,000,000), NIDDK antirat LH-S-11 (1:750,000), and antimouse TSH AFP98991 (1:300,000), respectively. Standard curves were constructed using the following standards: mouse PRL AFP10777D; mouse GH AFP10783B; rat LH, AFP11536B; and rat TSH, AFP11542B, respectively. The percentage bound in each assay was 32.3%, 46.3%, 20.5%, and 54%, respectively.

Magnetic resonance imaging (MRI)
Imaging of the animals was performed using a clinical whole body 1.5 T MR imaging system (Siemens Vision, Erlangen, Germany) at University Hospitals of Cleveland. All procedures were performed during nonclinical hours between 0600–0700 h, and all equipment was disinfected before and after animal imaging in compliance with the Department of Radiology Animal Research Policy. Animals were anesthetized by treatment with avertin and imaged using a small custom-built solenoidal receiver coil. We obtained T1-weighted spin echo images (repetition time, 400 msec; echo time, 14 msec; number of signal averages, 4; imaging time, 6 min, 53 sec; slice thickness, 1 mm; in-plane resolution, 195 µm) in the coronal and sagittal imaging planes. A computed tomography scan was performed in one animal to confirm the bony landmarks denoting the location of the sella turcica and pituitary gland. Pituitary volume was determined by multiplying pixel volume by the number of pixels within the pituitary gland as defined by a region of interest that was manually drawn on magnified sagittal images using the MR system console. All imaging was performed blind.

Statistical analyses
A two-tailed t test, assuming unequal variance, was performed for all studies, except for analysis of serum hormone levels. Circulating hormone levels were analyzed using SAS software version 8e (SAS Institute, Inc.A, Cary, NC). Multivariate regression models were used to determine the independent effects of age, the LH transgene, and ovariectomy status on the dependent variable. All combinations of interactions between the independent variables were included in the models. Means were compared using the LSMEANS function of PROC GLM. P values, representing differences between the group means, were adjusted using a Bonferroni correction to account for multiple comparisons. The distributions of several measures were found to be skewed toward the upper ranges. These variables were log-transformed before being analyzed. In addition, if during bivariate analyses, heteroscadacity of the dependent variable was observed across strata of the independent variable, significance tests for the differences in means were confirmed using t tests assuming unequal variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Elevated LH leads to an increase in pituitary size accompanied by selective hyperproliferation of Pit-1-positive cells
Transgenic females have elevated LH beginning as early as 2 wk of age (35). Elevated LH leads to chronic ovarian hyperstimulation, resulting in an increase in pituitary size in all strains of mice examined (progeny from crosses between CF1 LHCTP males with CF1, C57BL/6, and FVB/N females). Although the original studies were carried out in CF1, this strain does not survive past 5 months due to the appearance of ovarian tumors, followed by renal failure (34, 35). Thus, all mice used in the current studies were female progeny from crosses between LHCTP-CF1 males and FVB/N females (F1). In this cross, LH is elevated 5-fold at 16 wk of age (Fig. 1AGo).



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  		FIG. 1. Pituitary volume is increased at 16 wk in transgenic females with elevated LH. A, Serum LH was measured in wild-type (WT; n = 6) and transgenic (TG; n = 4) females at 16 wk. Values represent the mean ± SEM. *, P < 0.05. B, Representative sagittal MRI scans of a wild-type and a transgenic female at 16 wk of age. Arrows indicate the pituitary gland in both wild-type and transgenic animals. C, Pituitary volume was measured by MRI for wild-type (n = 5) and transgenic (n = 12) females at 16 wk of age. Values represent the mean ± SEM. *, P < 0.05, comparing transgenic volumes to those of wild-type animals.

 
An increase in the size of pituitaries in some transgenic females was evident by 8 wk of age (data not shown), and by 16 wk all transgenic females had enlarged pituitary glands when evaluated by MRI (Fig. 1BGo). In fact, pituitary volume was increased by approximately 2-fold in transgenic females relative to wild-type controls (4.9 ± 0.4 vs. 2.5 ± 0.6 mm3) by 16 wk of age (Fig. 1CGo).

To assess whether the increase in pituitary size was accompanied by an increase in proliferation, we performed immunohistochemical analyses of pituitary sections from 20- to 24-wk-old transgenic and wild-type females. Similar to the 16-wk-old group, the pituitary glands in the transgenic females were enlarged relative to those in age-matched wild-type controls (Fig. 3AGo). Initially, we performed immunohistochemistry using antibodies against the mitotic marker, PCNA (45, 46), to determine whether there was an increase in proliferation. There was an increase in the number of PCNA-positive cells in transgenics (data not shown); therefore, to identify which cells were proliferating, we performed dual immunohistochemistry using antibodies against Pit-1 and PCNA. These studies revealed a selective increase in the proliferation of Pit-1-positive cells in transgenic females (Fig. 2Go). All PCNA-positive cells in both wild-type and transgenic animals were also Pit-1 positive. The number of PCNA-positive cells per section was normalized to the total number of Pit-1-positive cells per field, and with this correction there was an approximately 10-fold increase in the number of PCNA-positive cells in transgenic glands relative to wild-type glands, indicating an increase in proliferation (Fig. 2BGo).



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  		FIG. 3. Hypertrophy of lactotropes and hyperplasia of somatotropes and thyrotropes is blocked by ovariectomy. Mice were OVX at 6 wk of age, and pituitary weight was measured for wild-type intact (n = 4), transgenic intact (n = 6), wild-type OVX (n = 5), and transgenic OVX (n = 7) females at 20 wk of age, immediately postmortem (A). Shown are representative sections from pituitaries of wild-type and transgenic females, both intact and OVX. Antibodies against PRL (B), GH (C), and TSH (D) were used in immunohistochemical analyses of pituitary sections. All insets represent propidium iodide stain of nuclei in the same field. E, Serum hormone levels were measured using specific RIA for wild-type (n = 6), transgenic (n = 10), wild-type OVX (n = 4), and transgenic OVX (n = 8) females. Values represent the mean ± SEM. *, P < 0.05 vs. wild-type; a, P < 0.05 vs. intact of the same genotype; b, P < 0.02, uncorrected, vs. WT intact.

 


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  		FIG. 2. Pit-1-positive cells are hyperproliferative in pituitary glands of transgenic females. A, Representative immunohistochemical analyses of pituitaries from wild-type (n = 5) and transgenic (n = 6) females. Antibodies against PCNA (red) and Pit-1 (green) were used in dual immunohistochemistry. The merged images of the same fields demonstrated colocalization (arrow) of all PCNA-positive cells with Pit-1-positive cells. The blue panel represents 4',6-diamido-2-phenylindole hydrochloride stain of all nuclei in each field. B, All PCNA-positive cells in the entire section (at least two sections per animal) were counted manually. The number of Pit-1-positive cells in at least two fields per section was quantitated with Metamorph. The total number of PCNA-positive cells per section was normalized to the average number of Pit-1-positive cells per field. Values represent the mean ± SEM. *, P < 0.05, comparing PCNA/Pit-1-positive cells in transgenic pituitaries to the number in wild-type animals.

 
Increase in pituitary size is due to lactotrope hypertrophy as well as somatotrope and thyrotrope hyperplasia
Although the PCNA labeling indicated that Pit-1 cells were hyperproliferative, it was unclear whether all three cell types of this lineage, lactotropes, somatotropes, and thyrotropes, were involved. Therefore, we examined by immunohistochemistry pituitary sections obtained from mice at 20–24 wk (Fig. 3Go, B and D), counted the number of hormone-positive cells, and normalized them to the total number of cells per field (Table 1Go). Consistent with the Pit-1 data, the percentages of corticotropes and gonadotropes (data not shown) remained unchanged when wild-type and transgenic pituitaries were compared. In contrast, somatotropes and thyrotropes were increased in transgenic pituitaries. Lactotropes were clearly hypertrophic, but we were unable to document lactotrope hyperplasia. Together, these data suggest that chronic ovarian hyperstimulation leads to the proliferation of all three types that comprise the Pit-1-dependent lineage.


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  		TABLE 1. GH- and TSH-positive cells are hyperplastic in transgenic females

 
We hypothesized that the pituitary pathology in LHCTP mice was the result of an indirect effect of LH on the pituitary gland. To determine whether the ovary was mediating the increase in pituitary size, transgenic mice were ovariectomized (OVX) at 6 wk of age and followed to 20 wk of age. Pituitary glands were weighed immediately postmortem at 20 wk. The transgenic glands weighed significantly more than the wild-type pituitaries, confirming the results of the earlier MRI scans (Fig. 3AGo). However, ovariectomy resulted in a reduction in pituitary weight only in the transgenic glands, suggesting that the increase in size depends on maintained chronic ovarian hyperstimulation. The pituitary glands from OVX mice were sectioned, and immunohistochemical analyses were performed as previously (Fig. 3Go, B–D, and Table 1Go). Lactotropes decreased in number and were no longer hypertrophic in animals OVX at 6 wk of age. Somatotropes and thyrotropes were also no longer hypertrophic in OVX transgenics and appeared to decrease in number; however, this was not statistically significant compared with intact transgenics. These data suggest that some alteration at the level of the pituitary gland or the hypothalamus has occurred before 6 wk of age and that the ovary is no longer required for maintenance of hyperplasia after 6 wk of age. Circulating levels of pituitary hormones were measured in intact animals to determine whether the morphological changes in the Pit-1-positive cells affected their function (Fig. 3EGo). In this regard, serum PRL and GH levels were elevated at 20–24 wk in transgenic females, indicating functional alteration of these cell types. In contrast, TSH levels were not appreciably increased despite the increased numbers of thyrotropes in transgenic females, and, as expected, FSH levels were unchanged. The maintenance of elevated levels of PRL and GH in transgenic mice is dependent on the ovary, as their levels at 20–24 wk of age were no longer elevated when ovariectomy was performed at 6 wk of age.

LHCTP females develop pituitary adenomas
To determine whether hyperproliferation of Pit-1-positive cells could lead to the development of pituitary adenomas, we evaluated LHCTP females at older ages. Figure 4AGo depicts the gross morphology of the pituitaries from a wild-type and transgenic female removed from the skull. The transgenic glands had regions of hemorrhaghia emanating from the anterior lobes. Pituitaries from transgenic animals as well as wild-type littermates were weighed immediately postmortem. Pituitary tumor weight was approximately 10-fold greater than that of pituitary glands from wild-type controls. Histological analysis of sections from tumors revealed a number of pathological markers common to pituitary adenomas (Fig. 4CGo). These include compression of the intermediate and posterior lobes. In addition, the morphology of the anterior lobe was disrupted by regions of peliosis, or dilated sinusoids, that appear as pools of blood without endothelial lining. This is a common feature of pituitary adenomas (46A ). The adenohypophysis was also marked with areas of hematoid deposition and mitotic figures.



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  		FIG. 4. Transgenic females develop numerous neoplastic lesions by 10 months. A, Transgenic glands (right) grossly resemble adenomas and remain significantly larger (n = 5) than wild-type controls (n = 4) at 10–12 months of age (B). Values represent the mean ± SEM. *, P < 0.05 vs. wild-type. C, A representative section of a wild-type pituitary gland stained with hematoxylin and eosin. D, Representative sections from transgenic glands are shown, and arrows point to compressed intermediate lobe, peliosis, mitotic figures, and hematoid depositions (top to bottom).

 
A hallmark of pituitary adenomas is the breakdown of reticulin fibers within the tumor (46A ). Generally, the area surrounding the tumor is defined by increased silver stain due to what appears to be a condensed border of reticulin. In contrast, diffuse hyperplasia shows no disruption of the reticulin network, and nodular hyperplasia is characterized by little, if any, compression, resulting in a seemingly thickened rim of acini (47). Gordon-Sweet silver staining of the reticulin network was performed to assess acinar structure in LHCTP mice. Figure 5AGo shows the normal acinar structure of a wild-type pituitary gland. In contrast, the reticulin fibers appear to be lost within the tumor, marking a loss of acinar structure (Fig. 5BGo). In addition, there is a classical condensation of reticulin fibers in the area surrounding the tumor (Fig. 5CGo). Thus, by 10 months of age, chronic ovarian hyperstimulation results in the development of adenomas in female mice.



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  		FIG. 5. Disruption of the reticulin network confirms the presence of adenomas. Shown are representative sections from wild-type and transgenic pituitaries silver-stained using the Gordon-Sweet method. A, Section from a wild-type pituitary gland. The arrow points to normal reticulin fibers demonstrating normal acinar structure (enlarged to the right). B, Field within a representative pituitary adenoma that contains no reticulin network (enlarged to the right). C, The arrow points to a condensed area of positively staining reticulin in the area surrounding a tumor, and the asterisk denotes a region of the tumor that does not contain any silver stain.

 
Pituitary tumors in LHCTP females are functioning adenomas containing multiple cell types
Immunohistochemical analyses of pituitary tumors collected from mice at 10–12 months of age was performed to assess which cell types were present within the adenomas (Fig. 6Go). All tumors exhibited positive staining for PRL. Figure 6AGo depicts a representative tumor focus containing PRL. Although all animals tested contained tumors positive for PRL, two tumors also contained foci of somatotropes, and three had thyrotrope foci (Fig. 6Go, B and C, respectively).



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  		FIG. 6. Pituitary tumors in LHCTP mice contain foci of lactotropes, somatotropes, and thyrotropes. Shown are representative sections from transgenic glands (n = 17) containing tumors (right panels) and age-matched wild-type (n = 6) littermates (left panels). Tumors contain foci of lactotropes (A), somatotropes (B), and thyrotropes (C). A, Anterior lobe; I, intermediate lobe; P, posterior lobe.

 
Measurement of circulating hormone levels suggests that the pituitary tumors in LHCTP females are functioning adenomas. Serum levels of PRL and GH are significantly higher in transgenic mice bearing tumors than in age-matched wild-type littermates (Fig. 7Go, A and B). Although TSH levels are not significantly different in these older animals (data not shown), there did appear to be a trend toward higher levels in transgenic females. Ovariectomy at 16 wk of age blocked the elevation in serum PRL and GH at 40 wk of age. These data suggest that LH overexpression acts to chronically hyperstimulate the ovary and will, through this indirect effect, culminate in the presence of functioning, hormone-secreting tumors.



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  		FIG. 7. Elevated circulating levels of pituitary hormones indicate that adenomas in LHCTP females are functional tumors. Serum levels of PRL (A) and GH (B) are significantly increased in transgenic females (*, P < 0.05 vs. WT) at 10–12 months of age. Ovariectomy was performed at 16 wk of age, and hormone levels were measured at 10 months of age. Ovariectomy at 16 wk of age reduced circulating levels of both hormones to levels not different from those in wild-type controls by 10 months of age (a, P < 0.05 vs. intact of the same genotype).

 
Ovariectomy followed by estradiol only partially recapitulates the LHCTP phenotype
It is clear that a hyperstimulated ovary is required for the development of pituitary hyperplasia in LHCTP females, as demonstrated by the ovariectomy studies in 6-wk-old mice. As shown by Milliken and colleagues (37), serum levels of steroid hormones, including estradiol, are increased as early as 5 wk of age and remain elevated through 41 wk of age. To test whether estrogen alone was sufficient for the development of pituitary tumors in LHCTP females, we OVX mice at 16 wk of age and replaced estradiol through 10 months of age. We chose to ovariectomize mice at 16 wk because pituitaries are already hyperplastic by this age and any continued influence from the ovary would probably be involved in maintaining hypertrophy and potential progression to adenoma, rather than initiation of these events.

Ovariectomy of transgenics at 16 wk resulted in a significant decrease in pituitary volume at 10 months of age, as measured by MRI. Estradiol replacement using sustained release pellets that contain 17ß-estradiol prevented this decrease in size (Fig. 8Go, A and B), and pituitary volume in these mice was not significantly different from that in transgenics that were subjected to sham surgery and treated with placebo. Immunohistochemical analyses of pituitary sections demonstrated a reduction in PRL staining of OVX transgenics, which was maintained by estradiol treatment (Fig. 8CGo). Ultimately, there were PRL-containing foci in both intact transgenics treated with placebo and transgenics that were OVX and treated with estradiol (Fig. 8FGo). However, no foci were observed in wild-type mice that were OVX and received estradiol, suggesting that the LHCTP pituitary has an altered responsiveness to the effects of estradiol. We were still able to see expansion of GH and TSH cells in some sections from intact, placebo-treated transgenics (Fig. 8Go, D and E). However, in OVX, estradiol-treated transgenics, we were unable to find any sections that contained a large number of cells other than lactotropes. Therefore, GH staining appeared reduced in representative sections from OVX transgenics and was even further decreased by estrogen replacement (Fig. 8DGo). This is most likely due to the presence of PRL-containing foci in these mice involving a possible trans-differentiation of somatotropes to lactotropes (48). Thus, the maintenance of GH-containing foci may require continual ovarian input. Similar results were observed for thyrotropes, and no TSH containing foci were present in OVX transgenic mice treated with estradiol (Fig. 8EGo). Therefore, the intact ovary and a combination of hormones are most likely required for maintaining adenoma development of all three cell types, whereas estradiol alone is only sufficient for prolactinoma formation in transgenic mice. These data suggest that although estradiol cannot initiate a tumor in wild-type mice, it promotes the formation of prolactinomas in transgenic mice that have received chronic ovarian input during the first 16 wk of life.



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  		FIG. 8. Ovariectomy with estradiol replacement restores prolactinoma development, but does not support hyperplasia of somatotropes and thyrotropes. Mice were OVX and treated with estradiol at 16 wk of age and killed at 10 months of age, at which time all measurements were taken. A, Representative sagittal MRI scans from intact/placebo (P)-treated transgenics, OVX/P transgenics, and OVX/estrogen (E)-treated transgenics in this order. Arrows point to the pituitary gland in each. B, Pituitary volume was measured for wild-type (WT) intact/P (n = 4), transgenic (TG) intact/P (n = 5), WT OVX/P (n = 6), TG OVX/P (n = 5), WT OVX/E (n = 3), and TG OVX/E (n = 3). Values represent the mean ± SEM. *, P < 0.05 vs. wild-type; a, P < 0.05 vs. intact of the same genotype. Sections from (left to right) intact/P transgenics, OVX/P transgenics, and OVX/E-treated transgenics were stained for PRL (C), GH (D), and TSH (E). Insets represent propidium iodide-stained nuclei of the same field. F, PRL-containing foci are seen only in transgenics (left) and OVX/E-treated transgenics (right).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Steadily emerging evidence indicates that a complex set of molecular events underlie the formation of pituitary adenomas. These events can be broadly categorized as either intrinsic pituitary defects or as extrinsic factors, such as hypothalamic hormones, steroids, and cytokines, that together may act to allow selective focal expansion of specific pituitary cell types (2, 3, 49). Herein we suggest that abnormally elevated levels of LH may also be regarded as a significant extrinsic factor that acts through the ovary to underlie the formation of functional adenomas restricted to cells of the Pit-1 lineage. Furthermore, our estradiol replacement studies indicate that chronic elevations in LH sensitize the murine pituitary gland to the tumorigenic effects of estradiol.

One of the unusual features of our transgenic model is the association of chronic elevations of LH with the formation of Pit1-containing adenomas. It seems likely that elevated LH acts directly through the ovary to indirectly affect the pituitary glands in LHCTP mice. Indeed, transgenic mice OVX at 6 wk of age fail to develop enlarged pituitaries. and mice that are OVX at 16 wk, an age at which Pit-1 cells are hyperproliferative, fail to develop adenomas. Because serum LH reaches even higher levels in OVX transgenic mice (33), the response of the pituitary in animals with an intact ovary most likely reflects the direct action of LH on this organ rather than on the pituitary. In the absence of an ovary, treatment with estradiol is able to promote the lactotrope phenotype in transgenic mice, but is not sufficient to support thyrotrope and somatotrope expansion. Consistent with our data, it is unlikely that estradiol alone would be sufficient to result in the appearance of tumor foci containing thyrotropes or somatotropes. Of note, wild-type mice OVX and treated with estradiol do not have enlarged pituitaries with PRL-containing foci, indicating that the effects of estradiol on prolactinoma development requires additional molecular events unique to the LHCTP mouse that may sensitize the pituitary to the effects of estradiol. This contrasts with studies carried out in Fischer 344 rats, which develop prolactinomas upon ovariectomy and treatment with diethylstilbestrol without additional insults, such as chronically elevated LH (30, 31). Thus, the data presented in this report suggest that critical molecular changes have occurred in the pituitary glands of LHCTP mice by 16 wk of age, altering the responsiveness of the pituitary to estradiol.

Restricted hyperproliferation of Pit-1-positive cells is another unique feature of our transgenic model. This restricted hyperplasia may reflect alterations involving multiple axes. As elevated LH causes hyperandrogenemia in female LHCTP transgenics, this steroid may contribute to the increase in somatotropes through the regulation of GHRH pulse frequency (35, 51, 52). Removal of the ovary would then cause a drastic reduction in testosterone, potentially eliminating a trophic factor for somatotropes, resulting in the loss of somatotrope transformation. In addition, Kero and colleagues (40, 53, 54) demonstrated that ectopic expression of LH receptors in the adrenal cortex of LHCTP females results in ovary-dependent, elevated glucocorticoid production. Of note, in addition to ACTH-secreting cells in the pituitary, glucocorticoids regulate GH-secreting cells in early differentiation of the somatotrope cell lineage and also increase both GH and GHRH receptor expression (55, 56, 57). Thus, due to indirect effects, it is possible that elevated glucocorticoids may drive the expansion of the GH-producing cells in our model. Conversely, the high levels of glucocorticoids may contribute to the restricted pituitary hyperplasia in LHCTP mice through the established negative effects on corticotrope proliferation (58). Similarly, the elevated levels of estrogen and androgens may be involved in preventing hyperplasia of gonadotropes. Ultimately, it is likely that the concerted effects of multiple hormones from both the ovary and the adrenal gland contribute to the restriction of the pituitary phenotype to three cell types in LHCTP mice.

Dysregulation of additional hypothalamic-derived factors may play a role in the increased proliferative status of Pit-1-positive cells in LHCTP pituitaries. For example, regulation of lactotrope proliferation as well as PRL synthesis is under tonic inhibition by dopamine, and disruptions to this axis result in lactotrope hyperplasia, hyperprolactinemia, and prolactinoma formation (9, 10, 59, 60, 61). Consequently, any alteration in dopamine or the sensitivity of the pituitary gland to the effects of this neurohormone may also contribute to hyperprolactinemia and prolactinoma development in LHCTP mice.

In addition to an increase in extrinsic factors, such as estrogen, it is likely that there are a number of intrinsic molecular changes that occur at the level of the pituitary gland in LHCTP mice. For example, this mechanism has been put forth in the case of estrogen-induced prolactinoma in F344 rats, in which treatment with diethylstilbestrol induces the expression of both basic fibroblast growth factor and pituitary tumor transforming gene (62). Similarly, in C57BL/6 mice, estrogen alone does not consistently give rise to pituitary tumors, but the combinatorial effects of this trophic factor with the loss of TGFß type II receptors results in adenoma development (63). Therefore, we are currently carrying out gene expression profiling studies using microarrays as well as breeding to additional mouse models to identify intrinsic changes that underlie the formation of adenomas of the Pit-1 lineage.

During the course of their studies, Rulli et al. (64) reported that transgenic mice expressing excessive levels of hCG develop ovarian-dependent hyperprolactinemia associated with the formation of lactotrope adenomas. It is unclear, however, whether somatotropes and thyrotropes exhibit hyperplasia and subsequent adenatomous formation as they do in our LHCTP transgenic model. If this discrepancy does exist, it may be attributed to the dramatically elevated levels of hCG that place the concentration of the hormone in the pharmacological range, whereas our LHCTP mice have only a 5- to 10-fold increase in LH (34), placing it in a pathophysiological, rather than pharmacological, range.

In summary, we have developed a unique model of chronic ovarian hyperstimulation that results in the development of pituitary adenomas containing multiple cell types. Supporting the utility of this model, in humans, chronically elevated LH has been described in women with polycystic ovary syndrome (41, 42). In addition, ovarian hyperstimulation with and without an increase in serum estradiol has been implicated in the development of at least one type of adenoma (65, 66). Our estradiol replacement data offer intriguing evidence that the pituitary glands of LHCTP females have an altered responsiveness to the transforming effects of estradiol, potentially providing a new murine model of estrogen-induced prolactinoma. Thus, LHCTP mice provide a new model for examining control of the proliferative status of Pit-1-positive cells and tumors that may contain foci of one of three cell types as well as understanding the mechanisms that may sensitize the pituitary to the transforming effects of estradiol.


   Acknowledgments
 
We acknowledge Mark Cohen for his assistance with histological analysis, Jonathan Mosley for statistical expertise, David Peck for animal husbandry, Simon Rhodes for the gift of a Pit-1 antibody, and Darcie Seachrist for Metamorph analysis and cell counting.


   Footnotes
 
This work was supported by NIH Grants DK-07319 (to R.A.A.), RO1-DK-28559 (to J.H.N.), and R01-CA-86387 (to J.H.N.).

1 Present address for R.A.A.: Division of Endocrinology, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, California 90048. Back

Abbreviations: FITC, Fluorescein isothiocyanate; MRI, magnetic resonance imaging; OVX, ovariectomized; PCNA, proliferating cell nuclear antigen.

Received March 24, 2003.

Accepted for publication July 7, 2003.


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 Results
 Discussion
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