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Endocrinology Vol. 143, No. 11 4429-4436
Copyright © 2002 by The Endocrine Society

ARTICLE

Pituitary Tumorigenesis in Prolactin Gene-Disrupted Mice

Martha E. Cruz-Soto, Michael D. Scheiber, Karen A. Gregerson, Gregory P. Boivin and Nelson D. Horseman

Molecular and Cellular Physiology (M.E.C.-S., N.D.H.), Departments of Obstetrics and Gynecology (M.D.S.) and Pathology and Laboratory Medicine (G.P.B.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Department of Pharmacological and Physiological Sciences, St. Louis University (K.A.G.), St. Louis, Missouri 63104

Address all correspondence and requests for reprints to: Nelson D. Horseman, Ph.D., Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576. E-mail: nelson.horseman{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Targeted disruption [knockout (KO)] of the mouse prolactin (PRL) gene created an animal model of primary isolated PRL deficiency in which there is no detectable PRL bioactivity. Pituitary glands of young adult female PRLKO mice were hyperplastic, and many cells had expanded cytoplasms with granular accumulations of an N-terminal peptide encoded by the disrupted PRL gene (KO/10 peptide). Confocal imaging showed that the pituitaries in PRL+/+ and PRL+/- females contained dense accumulations of apparently Golgi-associated immunoreactive PRL. PRLKO female mice (15–18 months old) developed hyperemic pituitary adenomas. The pituitary tumors in PRLKO mice synthesized the KO/10 peptide, which implies that the tumors arise from the lactotroph lineage. Anchorage-independent growth was observed among pituitary cells from PRLKO mice, aged 8 months or older, but not in cells from 3-month-old PRLKO mice. GH cells appeared to be normal in PRLKO pituitaries, but were displaced by the hyperplastic and hypertrophic growth of KO/10-positive cells. Bromocriptine suppressed mean pituitary weight in 8-month-old PRLKO mice compared with vehicle-treated PRLKO animals (20 ± 0.01 and 60 ± 10 mg; P < 0.01). We infer that pituitary lactotrophs of PRLKO mice suffer from a dual pathology that includes hypertrophy resulting from endoplasmic reticulum expansion and hyperplasia, with adenomatous transformation, in part as a consequence of disrupted dopaminergic feedback regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROENDOCRINE regulation of prolactin (PRL) cells (lactotrophs) in the anterior pituitary gland of mammals is unique. Unlike other mammalian adenohypophyseal cells and nonmammalian lactotrophs, for which secretory control is largely stimulatory, the control of mammalian lactotrophs is primarily inhibitory. Tuberoinfundibular dopaminergic (TIDA) neurons, with cell bodies in the arcuate nucleus of the hypothalamus (catecholaminergic area A12), project to the median eminence, where dopamine (DA) is secreted into the portal system and transported to the adenohypophysis (1, 2). DA activates type 2 (D2) DA receptors on lactotrophs in the anterior pituitary to inhibit PRL synthesis and secretion and lactotroph proliferation. The mechanisms by which DA suppresses lactotroph function may include both decreased adenylyl cyclase activity (3) and membrane hyperpolarization (4).

PRL elicits its negative feedback action by causing TIDA neurons to increase DA synthesis and secretion (5, 6, 7). This PRL effect is thought to be mediated in part by inducing tyrosine hydroxylase (TH), the rate-limiting enzymatic step in DA synthesis. Evidence for the role of TH in PRL feedback includes an increase in mRNA and TH activity in the hyperprolactinemic state as well as reduced TH activity and mRNA in experimentally produced hypoprolactinemia (8).

Pituitary adenomas are common human neoplasms. Autopsy and magnetic resonance imaging series estimate the incidence of pituitary adenomas to be approximately 20% in the general population (9, 10). Prolactinomas are the most common of these pituitary neoplasms. Prevailing theories of tumorigenesis suggest that a complex interaction between hormonal regulation and an intrinsic pituitary defect results in the neoplastic growth (11). Mice with a disrupted D2 DA receptor gene develop chronic hyperprolactinemia, lactotroph hyperplasia, and pituitary adenomas (12, 13), suggesting that loss of dopamine signaling, acting either directly or through elevation of PRL synthesis, leads to lactotroph hyperproliferation.

Targeted disruption of the PRL structural gene in mice has produced a novel model of isolated PRL deficiency (PRLKO) in which the complex physiological interactions of lactotrophs with their hypothalamic regulatory system may be studied (14). Previous studies in adult male PRLKO mice demonstrated an increase in pituitary weight associated with a concurrent specific reduction in DA content in the median eminence (15). These earlier studies did not characterize the cellular composition of the enlarged pituitaries. Gonadotropin release in vitro suggested that the gonadotroph population in PRLKO mice was unaffected and the growth of PRLKO mice was normal, indicating that GH and thyroid hormone status were substantially normal.

The present studies were undertaken to characterize the effect of PRL deficiency on the mouse adenohypophysis. These studies differ from earlier efforts in that we focused exclusively on female animals and designed our experiments to allow us to identify a cellular origin for the abnormal pituitary growth seen in PRLKO mice. In addition, treatment with a dopaminergic agonist allowed us to determine that abnormal dopaminergic input may be sufficient to explain the dysregulation of pituitary growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
PRLKO animals were produced by breeding PRLKO (PRL-/-) males with phenotypically normal (PRL+/-) females, and offspring were genotyped by PCR analysis of DNA obtained from tail biopsies (14). Animals were housed in a pathogen-free environment on a 14-h light, 10-h dark cycle and were fed a conventional diet of mouse ration and water ad libitum. Animals were killed by CO2 inhalation at selected ages between 6 wk and 17 months. The range of ages was selected considering two criteria: attainment of sexual maturity and age at which the first animal died, respectively. Tissues were harvested by sharp dissection of the brain and pituitary under a dissecting microscope. Tissue was fixed in 10% neutral buffered formalin, dehydrated through a gradient of alchohols, and embedded in paraffin. Tissue sections were cut at 4-µm thickness and stained with hematoxylin and eosin (H&E) (16). Animal protocols were approved by the institutional animal care and use committee.

Immunohistochemistry
The mouse PRL gene was disrupted by insertion of a neomycin resistance cassette in the middle of the coding region (14). This targeting strategy resulted in the encoding of a novel 10-amino-acid sequence (DPPGCRNSIS) at the C terminus of the disrupted PRL gene, and the synthesis of an approximately 10-kDa unique peptide (PRLKO/10) in mice carrying the PRLKO allele (14). An antibody, designated anti-KO/10, was generated in rabbits to the epitope at the C terminus of KO/10. A second antibody to the native PRL protein (anti-PRL/C25) was generated to a peptide sequence of the mouse PRL gene C terminus (LRRDSHKVDNFLKVLRCQIAHQNNC). Because the mRNA transcribed from the disrupted PRL allele truncates before sequence encoding the native C terminus, the C/25 peptide was absent from the KO/10 peptide expressed in PRLKO mice. Each respective peptide was conjugated with keyhole limpet hemocyanin and used to immunize rabbits. Serial bleedings were performed on these rabbits, and after sufficient titers were obtained, antibodies were isolated and purified by peptide affinity chromatography as previously described (17).

For confocal immunofluorescence, sections of pituitaries were deparaffinized with xylene and rehydrated in a graded series of ethanol. Saponin (0.05%; 30 min) was used to permeabilize the tissue sections. Nonspecific binding was blocked by preincubation with normal goat serum (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA). The anti-KO/10 and anti-PRL/C25 antibodies were used at a 1:50 dilution in Tris-buffered saline/Tween (TBST). The anti-GH antiserum (gift from A. Parlow) was used at a 1:10,000 dilution in TBST. The protocol for the Vectastain kit was followed through incubation with biotinylated secondary antibody. At that point a 30-min incubation with Cy-3-conjugated avidin complex (Zymed Laboratories, Inc., San Francisco, CA; 1:1,000) in TBST was used for fluorescence staining. Sections were washed in PBS and incubated for 15 min with YO-PRO (Molecular Probes, Inc., Eugene, OR; 1:10,000) in PBS to stain the nuclei. After washing the sections in PBS and then in water, they were mounted in Aqua Poly/Mount (Polysciences, Inc., Warrington, PA). Images were produced on an LSM510 confocal microscope (Carl Zeiss, New York, NY) at the Microscopy and Imaging Core of the Cell Biology, Neurobiology, and Anatomy Department (University of Cincinnati). All confocal images were made using identical parameters (scan mode, scale, average, and pinhole).

Anchorage-independent growth assay
Pituitary glands were dissected from normal control (3-, 8-, and 12-month-old) and PRLKO (3-, 8-, 12-, and 15-month-old) mice and were washed three times with sterile PBS. They were minced and incubated with trypsin (0.25%) solution for 30 min at 37 C. The dispersed cells were diluted in DF medium (45% DMEM, 45% Ham’s F-12, and 10% fetal calf serum; Harlan Bioproducts for Science, Inc., Madison, WI), and washed by centrifugation. The washed cells were resuspended in DF medium, filtered through nylon mesh, and plated in six-chambered multiwell culture dishes. The culture medium was changed weekly, and when the cells had grown to confluence they were subcultured at a ratio of 1:3. Aliquots of cells were frozen at the second passage in 10% dimethylsulfoxide-containing cell freezing medium (Life Technologies, Inc., Gaithersburg, MD) for further analysis.

Confluent cells were dispersed by trypsinization and transferred to complete DF medium containing 0.4% Noble agar at a density of 1.8 x 106/100-mm tissue culture plate. Medium was replenished weekly, and colony formation was evaluated after 40 d in soft agar by direct visualization and vital staining with 3-[4,5-dimethylthiazol-2-yl]-1,5-diphenyltetrazolium bromide (Sigma, St. Louis, MO) according to the manufacturer’s instructions.

Serum estrogen assay
Random cycling, age-matched (4-month-old), normal (n = 6) and PRLKO (n = 7) female mice were used. Blood samples were collected by cardiac puncture while mice were under isoflurane anesthesia [IsoFlo (Abbot Laboratories, North Chicago, IL)]. Blood was allowed to clot overnight at 4 C. Serum was separated by centrifugation, transferred to microfuge tubes, and stored at -20 C until assayed. Unextracted serum was assayed using an estradiol ELISA kit (Estradiol EIA, DSL-10-4300) from Diagnostic Systems Laboratories, Inc. (Webster, TX) according to the manufacturer’s instructions.

Bromocriptine experiment
Pituitary tumors are seen in all PRLKO females 8 months or older; therefore, for this study we used female 8-month-old PRLKO mice. Animals were housed and cared for as described above. Daily sc injections of either bromocriptine mesylate (2.5 mg/kg; Sigma; 0.1 g bromocriptine dissolved in a minimum volume of absolute ethanol and suspended in a sesame oil vehicle to a total volume of 123 ml) or identical vehicle only was administered between 0800 and 1000 h daily for 4 wk. Animals were killed by CO2 inhalation, and pituitaries were removed as described above. Anterior pituitaries were blotted dry on absorbent tissue paper, weighed on an analytic scale, and fixed as described above for immunostaining.

Statistical analyses
Mean and SE were calculated for the pituitary weights. One-way ANOVA was used to test for differences among groups, and Tukey’s test was used to determine differences between treatments. A t test (two tailed) was used to test differences between two means. Significant differences were defined as P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gross pathology in aged (15- to 18-month-old) PRLKO mice
Early in the course of our experimentation we noted that pituitaries of all PRLKO females were enlarged by 6–8 wk of life, and pituitary hyperplasia was noted in PRLKO male mice (15). Subsequently, female PRLKO mice (n = 6) were kept in the laboratory to determine whether any disease processes attributable to disruption of the PRL gene were observed during their life span. At an age of 17 months, one mouse died, and another became anorectic and frail. The five aged mice remaining alive were killed and examined for gross pathologies. All of the aged PRLKO mice (15–18 months old) had developed large pituitary tumors (Fig. 1Go). As previously reported for young adult mice (14), multiple organs were examined histologically. No pathologies or lesions other than the pituitary adenomas noted here were observed in the aged (15- to 18-month-old) mice. The observation that pituitary tumors were the only observable lifetime pathology (aside from female infertility) (14) in laboratory-housed PRLKO mice provoked us to undertake a further examination of the biology of the PRLKO pituitary glands.



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  		Figure 1. Gross pathology of pituitaries from different genotypes. Left, Specimen from a normal mouse (age 15 months at death). Right, Specimen from a PRLKO mouse (age 15 months at death). Note the obvious enlargement of the pituitary from the KO mouse, consistent with the development of adenoma and vascular congestion.

 
Histology of normal and PRLKO pituitary glands
Histological findings are depicted in Fig. 2Go. H&E staining of pituitaries from at least three mice each in the PRL+/+ (6–8 wk), PRL+/- (6–8 wk), and PRL-/- (6–8 wk and 3, 6, 8, and 15 months) groups were used for these analyses, and the results shown are representative for each group. PRL+/+ and the heterozygous animals (Fig. 2Go, A and B) demonstrated normal pituitary architectures. The expected distribution of pituitary cell types in female animals was seen, with 50–60% eosinophilic cells (i.e. somatotrophs plus lactotrophs). Pituitaries from 6- to 8-wk-old PRLKO mice (Fig. 2CGo) had many cells with irregular boundaries and increased cytoplasmic volumes. Many cells contained large, weakly staining cytoplasmic inclusions. PRLKO mice that were 8 months or older consistently had large hyperemic pituitary adenomas, which were composed of monomorphic, weakly eosinophilic cells with large perinuclear cytoplasmic inclusions (Fig. 2DGo). Pools of blood cells were evident throughout the sections from the adenomas.



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  		Figure 2. Pituitary histology of the murine model of primary PRL deficiency. A, H&E-stained PRL+/+ pituitary. B, H&E-stained PRL+/- pituitary. C, H&E-stained PRL-/- hyperplastic pituitary (6 wk). D, H&E-stained PRLKO tumor pituitary (mouse aged 15 months). Objective lens magnification, x60/0.90 UPlanApo. Scale bar, 20 µm.

 
Novel antibodies that we generated were used to determine the cell line of origin that accounted for the hypertrophic, hyperplastic (6 wk or older), and adenomatous (8 months or older) pituitary growth in the PRLKO mice. Pituitary cells from either PRL+/+ (not shown) or PRL+/- mice (Fig. 3Go, D–F) stained with anti-PRL/C25 and showed discrete cytoplasmic accumulations of immunoreactive PRL. This localization is consistent with previous descriptions of normal production, storage, and release of PRL from the pituitary (18). Pituitary cells from PRL+/- mice had a punctate cytoplasmic staining with anti-KO/10 (Fig. 3Go, G–I). Our previous studies showed that heterozygous females synthesized the KO/10 peptide (14). These results indicate that the truncated peptide is expressed from the disrupted allele and is apparently not packaged in discrete granules along with native PRL. Further studies would be necessary to determine the release and/or degradation pathways for this peptide. Cells from hyperplastic (6 wk or older) PRLKO pituitaries displayed punctate cytoplasmic staining with anti-KO/10 (Fig. 3Go, J–L), and no staining with either preabsorbed anti-KO/10 or with anti-PRL/C25 (not shown). KO/10 immunoreactivity in pituitary tumors of aged (15- to 18-month-old) PRLKO mice did not have the punctate distribution that characterized KO/10 immunoreactivity in the younger specimen (compare Fig. 3Go, M–O with J–L), showing the predominance of cells from a lactotroph lineage within the tumor and the loss of discrete, punctate staining of KO/10. Using tumor sections, the controls for preabsorption with KO/10 peptide and anti-PRL/C25 were completely negative (not shown).



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  		Figure 3. Confocal immunofluorescence of female mouse pituitaries using anti-KO/10 and anti-PRL/C25. Each set of images is representative of staining patterns seen in specimens from at least three independently examined mice of the respective groups. Nuclei are stained in green (Yo-Pro), and immunopositive material in red (Cy-3). A–C, Negative control sections without primary antiserum. D–F, Staining of PRL+/- pituitaries using anti-PRL/C25. G–I, PRL+/- pituitaries with primary anti-KO/10. J–L, PRL-/- (3 m) hyperplastic pituitaries with anti-KO/10. M–O, PRLKO (15 months) tumor sections with anti-KO/10. B, E, H, K, and N, The green channel. C, F, I, L, and O, The red channel. A, D, G, J, and M, An enlargement (x10) of combined green and red channels. Note the differences between the consolidated deposits of PRL in D and F, the punctate and widely distributed deposits of KO/10 in G and I, and J and L, and the diffusely distributed KO/10 peptide in M and O. Objective lens magnification, x63/1.4 oil corr.c-apochromat. Scale bar, 20 µm.

 
GH cells in PRLKO mice
GH immunostaining was compared in PRL+/- and PRLKO mice to determine the effects of the changes in PRLKO pituitaries on a population of nonlactotroph cells (Fig. 4Go). Body growth was not significantly different in PRLKO mice (14), which suggested that somatotroph function was normal in these animals. It is possible that growth is affected at several months of age when tumors grow large enough to cause nonspecific effects because of the tumor mass. Indeed, somatotrophs appeared to be similar in normal and PRLKO mice, and a majority of GH-positive cells displayed the characteristic round morphology with circumferential GH immunostaining (Fig. 4Go, A and B), which was distinctly different from the immunostaining pattern of PRL-positive cells in the normal mice (Fig. 3Go, D and F).



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  		Figure 4. Confocal images of GH staining. A, C, and D, Sections from PRL+/- pituitaries. B, E, and F, Sections from PRLKO (3-month-old) hyperplastic pituitary; G and H, sections of PRLKO (15-month-old) tumor pituitary. A and B, Immunoreactive staining (x63/1.4 oil Dic plan-apochromat). C, E, and G, Lower magnification (x10/0.3 plan neofluar) of the positive staining of the PRL+/- pituitary, the PRLKO hyperplastic pituitary, and the PRLKO tumor pituitary sections, respectively. D, F, and H, The combined green and red channels of the same three sections. Each set of images is representative of staining patterns seen in specimens from at least three independently examined mice in the respective groups.

 
In the normal PRL+/- pituitary, GH cells were widely distributed, and there were no areas from which GH cells were excluded (Fig. 4Go, C and D). In contrast, GH cells were absent from large areas in hyperplastic (3-month-old) PRLKO pituitaries (Fig. 4Go, E and F) and were even more displaced in tumor sections from aged (15-month-old) PRLKO mice (Fig. 4Go, G and H).

Serum estrogens
Estrogen concentrations in unextracted serum were significantly lower in PRLKO females (41.2 pg/ml; 0.15 nM as 17ß-estradiol) compared with normal age-matched controls (73.1 pg/ml; 0.27 nM; P < 0.001, by t test). Similar estrogen levels were observed in a comparison of PRL receptor knockout and wild-type female mice, with a significant reduction in mice lacking the PRL receptor (19).

Anchorage-independent growth of PRLKO tumor cells
Pituitary cells cultured from mice 8 months and older grew as colonies in soft agar (Fig. 5Go, B and C), but those from 3-month-old mice remained as single cells (Fig. 5AGo). No colonies were seen in age-matched heterozygous animals. The calculated colony efficiency is 46%, which represents the number of colonies divided by the number of plated cells (x100%) (20). Consequently, we conclude that an anchorage-independent growth phenotype is acquired between 3 and 8 months in all PRLKO females.



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  		Figure 5. Anchorage-independent growth assay. A and B show pituitary PRLKO cells and colonies, respectively, stained with 3-[4,5-dimethylthiazol-2-yl]-1,5-diphenyl-tetrazolium bromide after 40 d of incubation. C shows unstained colonies under phase contrast. Single cells are seen in cultures from 3-month-old mice (A), whereas colonies are seen in cultures from 8-month-old mice (B and C). Objective lens magnification, x20/0.35. D shows a summary of all of the soft agar assays. Three mice from each age and from each genotype were used (n = 24). No colonies were found at any age in the PRL+/- mice. Colonies were found in one PRLKO animal by 6 months of age, whereas colonies were found in all PRLKO animals 8 months or older. Primary culture cells and cells from three or four passages were used for both genotypes. Each assay was performed in triplicate.

 
Bromocriptine therapy
A total of 26 animals were studied (PRLKO control, n = 7; PRLKO vehicle-only, n = 9; PRLKO bromocriptine-treated mice, n = 10) to determine the effect of a dopamine agonist on the PRLKO pituitary gland. Because pituitary hyperplasia and transformation were age related, we took care that the ages at death for the three groups were not different. Bromocriptine treatment had a significant effect on pituitary weight among the treated mice (by ANOVA: F = 8.7; df = 17; P < 0.01). Mean pituitary weight in PRLKO controls was significantly different from that in PRLKO treated animals (60 ± 0.01 and 20 ± 0.01 mg; Fig. 6AGo). Age-matched PRL+/- female animals (n = 4) had a mean pituitary weight of 4 mg (data not shown).



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  		Figure 6. Bromocriptine treatment reduces PRLKO tumor size. A, The wet anterior pituitary weights (in milligrams) of the three groups subjected to study: PRLKO animals that served as untreated controls, PRLKO animals that received vehicle only, and PRLKO animals that received bromocriptine treatment for 28 d. All animals were females and were 8 months old at the beginning of the treatment. B, Representative H&E-stained section of the pituitaries of animals that received vehicle only. C, Representative H&E-stained section of the pituitaries of animals that received bromocriptine treatment. Scale bar, 20 µm (x60/0.90 UPlanApo).

 
Immunohistochemical results from the pituitaries of animals in the bromocriptine experiment are depicted in Fig. 6Go, B and C. Pituitaries of the PRLKO vehicle control mice had many cells with expansive vesiculated cytoplasms (Fig. 6BGo). In marked contrast, bromocriptine therapy resulted in tumor regression (Fig. 6CGo) and cell shrinkage, as evidenced by pituitary cells in which the cytoplasm was less expanded and resembled the heterozygous controls (compare with Fig. 2BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Targeted disruption of the PRL gene results in a hypoprolactinemic phenotype that we have previously shown to cause female infertility and defective mammary gland development (14, 21). Although an extraordinary variety of biological effects have been ascribed to PRL (reviews in Refs. 22, 23, 24), PRL or PRL receptor deficiency does not result in major pathological changes in any tissues other than the female reproductive system and the pituitary gland (14, 25). These observations reinforce the concept that PRL is most important when homeostasis is challenged by circumstances such as reproduction and stress (26, 27), but is not essential during normal growth and steady state metabolism.

The present study demonstrates that PRLKO female mice develop a progression from pituitary hyperplasia (observed by 6 wk of age when sexual maturity has been reached) to frank pituitary adenomas (8 months or older), as defined with histopathological criteria and anchorage-independent growth. These findings in female animals extend earlier observations that gross pituitary weights were increased in male PRLKO mice (15). The cells that produce hyperplasia and adenomas in the pituitaries of PRLKO mice can be considered pseudolactotrophs, which were specifically identifiable based on their synthesis of KO/10 peptide from the disrupted PRL gene. The peptide expressed from the disrupted PRL gene is missing the critical structures that are responsible for PRL receptor binding, and we have shown that this peptide has no PRL bioactivity (14).

The cytoplasm of cells in the pituitaries of normal heterozygous mice stained intensely with anti-PRL/C25 and showed punctate staining with anti-KO/10. Native PRL is concentrated in the Golgi apparatus and secretory granules in the mouse pituitary (28). However, the KO/10 peptide is apparently not processed normally through the Golgi pathway into discreet secretory bodies.

Our immunohistochemical results in the hyperplastic pituitaries of PRLKO females (from 6 wk to 8 months of age) and the tumors from aged PRLKO females (8 months or older), both of which stain for anti-KO/10, demonstrated that the lactotroph is the cell of origin for tumors in PRLKO mice even though these cells do not produce bioactive PRL. The transformation of the lactotroph lineage in PRLKO mice argues against any indispensable role for PRL in the autocrine stimulation of prolactinomas.

Both male and female PRLKO mice undergo pituitary hyperplasia (Ref. 15 and the present study); however, the growth and eventual transformation of pseudolactotrophs appear to be accelerated in females compared with males. Because estrogen is a trophic factor for pituitary lactotrophs, and PRL is generally understood to suppress gonadal function (29, 30), we measured serum estrogen levels in normal and PRLKO mice. Rather than elevated estrogen levels, PRLKO mice had lower (by 44%) estrogen levels than those in the normal animals. Therefore, pituitary tumorigenesis in PRLKO mice cannot be explained by high estrogen levels, but may reflect an increased sensitivity to estrogens. D2 receptor-deficient mice have relatively low estrogen levels, and they still show sexual dimorphism in lactotroph hyperplasia (13). Another potential contributor to the sexual dimorphism in lactotroph growth is the pituitary neuropeptide galanin, which has been shown to stimulate lactotroph proliferation (31). Mice that are deficient in galanin have suppressed PRL levels and reduced numbers of lactotrophs (32).

The proposed mechanism of transformation of pituitary cells in PRLKO mice is through a loss of negative dopaminergic growth control, which results from a lack of PRL action on the hypothalamus. This is supported by the reduced DA content in the median eminence in male PRLKO mice (15). Hypothalamic dopaminergic neurons have PRL receptors, and these receptors may mediate a stimulatory effect on DA synthesis by virtue of activation of TH gene expression (8, 33, 34). TIDA neurons of PRLKO mice have reduced levels of both DA and TH (35), indicating that PRL deficiency leads to a deficit in DA biosynthesis in neurons that are involved in the feedback regulation of PRL synthesis.

Loss of dopaminergic growth control in PRLKO mice may have two effects. Firstly, cellular hypertrophy may result from the lack of transit of the truncated peptide through the endoplasmic reticulum (ER), causing an increase in ER size. Therefore, PRLKO cells adapt their size to the enlarged ER (36). This is evidenced in Figs. 2Go and 3Go, where the pseudolactotrophs show a clear increase in cell volume. Secondly, pseudolactotroph proliferation results in hyperplasia because of the imbalance created between the actions of positive growth regulators, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, and estrogen (37), and negative growth regulators, especially DA. This augmented proliferation may increase the statistical possibility of transforming mutations in daughter cells. Insufficient dopaminergic input to the pituitary allows for the accelerated clonal expansion of cells that have accumulated one or more transforming mutations. Thus, loss of DA tone may be a primary defect, leading to excess growth of cells that have not yet transformed, and a secondary defect, allowing proliferation of transformed cells.

Our current data, in accordance with previous ultrastructural morphometric electron microscopy findings (38, 39), agree with the morphology of bromocriptine-responding pituitary tumors before treatment and show that bromocriptine causes tumor regression, cell shrinkage, and irregular nuclear shape. The bromocriptine response of PRLKO cells supports the hypothesis that a lack of dopaminergic tone can contribute to pituitary tumorigenesis.

PRL has been shown to act as an autocrine growth factor for pituitary cells in culture (40) and has been suggested to subserve a similar function in vivo under some circumstances. D2 receptor knockout mice secrete high levels of PRL, as would be expected, and the correlation between high PRL levels and lactotroph hyperplasia led those who studied D2 receptor knockout mice to speculate that autocrine stimulation by PRL was responsible for lactotroph growth in their mice (12, 13). Contrary to this interpretation, our results show that bioactive PRL is not necessary for growth and transformation of cells of the lactotroph lineage. The pathology and temporal pattern of pituitary tumors in our mice are not substantially different from those in D2 receptor KO mice. Therefore, it appears that the lack of DA signaling alone, as a result of either inactivity of D2 receptors or inactivity of TIDA neurons because of low PRL, is sufficient to explain dysregulation of the lactotroph lineage in these mouse models.


   Acknowledgments
 
The authors acknowledge Stacy Shipman for her contributions to the initial observations in these studies. Jason Lockefeer, Kathryn Nieport, Matthew Herbst, and the technicians in the Division of Comparative Pathology contributed technical expertise to this project. The Microscopy and Imaging Core of the Cell Biology, Neurobiology, and Anatomy Departments at University of Cincinnati, especially Drs. R. Hennigan, N. Kleene, and C. Stamper added training and expert advice with the confocal microscope. Dr. A. Parlow from the NIDDK generously donated the pituitary GH antiserum.


   Footnotes
 
This work was supported by research grants and fellowships from the NIH (to N.D.H. and M.D.S.), a Fulbright Fellowship (National Council of Sciences and Technology of Mexico CONACyT/Garcia-Robles; to M.E.C.-S.), and a grant from Shriner’s Hospital for Children (to N.D.H.).

Abbreviations: DA, Dopamine; ER, endoplasmic reticulum; H&E, hematoxylin and eosin; PRL, prolactin; PRLKO, PRL knockout; TBST, Tris-buffered saline/Tween; TH, tyrosine hydroxylase; TIDA, tuberoinfundibular dopaminergic.

Received February 12, 2002.

Accepted for publication July 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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