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Endocrinology Vol. 141, No. 1 81-90
Copyright © 2000 by The Endocrine Society

ARTICLES

Pituitary Hormone Gene Expression and Secretion in Human Growth Hormone-Releasing Hormone Transgenic Mice: Focus on Lactotroph Function1

Joseph P. Moore, Jr.2, Aihua Cai, Mary Ellen Hostettler, Lydia A. Arbogast, James L. Voogt and James F. Hyde

Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky 40536; the Department of Physiology, Southern Illinois University School of Medicine (L.A.A.), Carbondale, Illinois 62901; the Department of Molecular and Integrative Physiology, University of Kansas Medical Center (J.L.V.), Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: James F. Hyde, Ph.D., Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, 800 Rose Street (MN224), Lexington, Kentucky 40536-0084. E-mail: jfhyde00{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per µg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSGENIC MICE overexpressing human GH-releasing hormone (hGHRH) show marked increases in circulating concentrations of endogenous GH and in body weight (1, 2). Moreover, hGHRH transgenic mice develop a progressive hyperplasia of the anterior pituitary gland that ultimately results in the formation of an adenoma (2, 3). The pituitary adenomas in older hGHRH transgenic mice consist primarily of somatotrophs and mammosomatotrophs (4, 5). Our characterization of the development of pituitary hyperplasia in hGHRH transgenic mice revealed an indiscriminate increase in the number of each of the primary cell phenotypes (6). However, the numbers of thyrotrophs, gonadotrophs, and corticotrophs within the hyperplastic pituitaries of the hGHRH transgenic mice were only modestly increased compared with the increased numbers of somatotrophs and lactotrophs.

The overexpression of hGHRH also results in alterations in the expression of regulatory peptides within the hypothalamus of the transgenic mice. Somatostatin (SS) messenger RNA (mRNA) levels are markedly increased within the hypothalamus of the hGHRH transgenic mouse, whereas mGHRH mRNA levels are significantly decreased (7, 8). These changes in the expression of neuropeptides are presumably the result of the elevated circulating levels of endogenous GH within the hGHRH transgenic mice. Conversely, the Ames dwarf mouse is deficient in GH (9). The hypothalami of these mice have significantly elevated levels of GHRH mRNA and peptide and decreased levels of SS mRNA and peptide (10, 11, 12).

Ames dwarf mice are also deficient in the anterior pituitary hormone PRL. The primary inhibitory regulator of PRL production and release is the catecholamine dopamine (13). Coincident with the PRL deficiency in Ames dwarf mice, dopamine levels and tyrosine hydroxylase (TH) expression in the tuberoinfundibular dopaminergic neuronal population are reduced (14, 15). In contrast, elevated levels of PRL are correlated with increased activity of tuberoinfundibular dopaminergic neurons (16). These animal models of extreme GH/PRL excess and deficiency demonstrate the magnitude of the influence that a pituitary hormone generates toward regulating the expression of neuroendocrine factors at the level of the hypothalamus.

Our recent studies revealed a 2-fold increase in the total number of lactotrophs in hGHRH transgenic mice (6), yet plasma levels of PRL are not elevated in hGHRH transgenic mice (17). The following studies were performed to explore the potential cellular neuroendocrine mechanisms underlying this disparity and to delineate whether gene expression or secretion of other pituitary hormones were similarly impacted. The objectives of this study were to 1) examine the relative differences in pituitary hormone gene expression and secretion in hGHRH transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell and in a static incubation system, 3) determine whether tyrosine hydroxylase gene expression and/or activity is altered in the hypothalamus of hGHRH transgenic mice, and 4) assess dopamine receptor gene expression, localization, and function in the anterior pituitary gland of hGHRH transgenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Dr. L. A. Frohman (University of Illinois-Chicago) supplied us with one male hGHRH transgenic mouse (from line 765–2), produced and described by Hammer et al. (1). These transgenic mice express a mouse metallathionein-1/hGHRH fusion gene. The single transgenic mouse was bred with female C57BL/6S mice (The Jackson Laboratory, Bar Harbor, ME). All mice were housed under controlled temperature and lighting (14 h of light) conditions and were provided water and laboratory chow ad libitum. The male mice were used at 4–6 months of age. The animals were rapidly anesthetized with ether and killed by decapitation for all tissue preparations. The hGHRH transgenic mice were identified by means of the PCR as described previously (18). All procedures involving animals were reviewed and approved by the institutional animal care and use committee at the University of Kentucky.

Tissue extraction for hormone content
Trunk blood was collected in heparinized microcentrifuge tubes, and the plasma was separated by centrifugation and stored at -20 C until analyzed for pituitary hormone content. Immediately after decapitation, the anterior pituitary gland was dissected and frozen on dry ice. The pituitaries were sonicated in 0.5 ml ice-cold 1.0 M acetic acid. The tissue homogenates were placed in a boiling bath for 10 min and then centrifuged at 10,000 x g for 10 min. Aliquots of the supernates were used to quantify protein contents by the method of Bradford (19). The supernates were then lyophilized and stored at -20 C until assayed in triplicate for hormone content.

Hormone determinations
Immunoreactive mouse PRL levels were determined in triplicate using a RIA kit provided by Dr. A. F. Parlow (Harbor-University of California-Los Angeles Medical Center, Torrance, CA). Highly purified mouse PRL (AFP-6476-C) was used as the reference preparation, highly purified mouse PRL (AFP-6476-C) was used for iodinated tracer, and a rabbit-generated antibody to mouse PRL (AFP-131078; 1:175,000 final dilution) was used as the primary antibody. Either 2.5% protein A or goat antirabbit IgG (1:8) was used to separate free from bound hormone. The sensitivity of the assay was less than 0.05 ng/tube.

Immunoreactive GH levels were determined by RIA, using materials provided by Dr. A. F. Parlow. Highly purified mouse GH (AFP-10783B) was used for iodination. Mouse GH (AFP-10783B) was used for the reference preparation, and a monkey-generated antibody to rat GH (NIDDK anti-rGH-S-5; 1:30,000 final dilution) was used as the primary antibody. Either 2.5% protein A or goat antimonkey IgG (1:6; Antibodies, Inc., Davis, CA) was used to separate free from bound hormone. The sensitivity of the assay was less than 0.05 ng/tube.

The mouse LH RIA used highly purified rat LH (NIDDK rLH-I-7) for iodinated tracer, rabbit-generated rat LH antiserum (NIDDK anti-rLH-S-10; 1:180,000 final dilution), and rat LH reference preparation (NIDDK rLH-RP-3) for standards. Bound antigen was separated from free antigen by precipitation with goat antirabbit {gamma}-globulins. The sensitivity of the assay was less than 5 pg/tube.

The mouse TSH RIA used highly purified rat TSH (NIDDK rTSH-I-9) for iodinated tracer, guinea pig-generated mouse TSH antiserum (AFP98991; 1:150,000 final dilution), and mouse TSH reference preparation (AFP51718MP) for standards. Bound antigen was separated from free antigen by precipitation with goat antiguinea pig {gamma}-globulins (Antibodies, Inc.). The sensitivity of the assay was less than 5 ng/tube.

Pituitary hormones were iodinated using either chloramine-T (Sigma, St. Louis, MO) or Iodogen (Pierce Chemical Co., Rockford, IL). Iodinated hormone was separated from free 125-iodine by passage over a Bio-Gel P-60 column (50–100 mesh; Bio-Rad Laboratories, Inc., Richmond, CA). The between- and within-assay coefficients of variation for all of the RIAs were less than 10%.

NSD-1015 injection and tissue preparation for HPLC
Transgenic and nontransgenic animals were injected ip with the 3,4- dihydroxyphenylalanine (DOPA) decarboxylase inhibitor NSD-1015 (100 mg/kg; Sigma) and returned to their cages. After 40 min, the animals were rapidly anesthetized with ether and killed by decapitation. The stalk-median eminence (SME) of each animal was carefully dissected with the aid of a dissection microscope and immediately snap-frozen on dry ice. The SME was sonicated in 0.1 N perchloric acid and centrifuged at 10,000 x g for 2 min. The pellets were solubilized in 0.5 N sodium hydroxide, and an aliquot was analyzed for protein content by the method of Bradford. The supernates were analyzed for DOPA concentrations by HPLC (CR4A; Shimadzu, Kyoto, Japan) equipped with a reverse phase ODS-5 column (16). The effluent from the column was passed through an electrochemical detector (LC-4B, Bioanalytical Systems, Inc., West Lafayette, IN) set at +0.65 V and 2 nA. The limit of detection was 20 pg. The amount of DOPA was quantified with a built-in integrator by comparing the peak area of the unknown sample with the peak areas of known amounts of standards.

RT-PCR
Mice were killed by decapitation, and the dissected tissues were rapidly frozen in liquid nitrogen. Total RNA was extracted according to the method of Chomczynski and Sacchi (20). RT-PCR was performed using the GeneAmp PCR kit (Perkin-Elmer Corp., Branchburg, NJ) following the instructions of the manufacturer with the addition of {alpha}-32P-labeled deoxy-CTP. Table 1Go is a list of the specific oligonucleotides used as primer sets for PCR (Integrated DNA Technologies, Inc., Coralville, IA). The PCR reactions were run at 95 C for 1 min, 60 C for 1 min, and 72 C for 2 min. Preliminary experiments were performed to optimize the PCR conditions. Relative differences in gene expression were determined after optimizing the following PCR conditions. 1) The number of cycles for each primer set was carefully selected such that the PCR products were within their linear range (GH and PRL- 20 cycles; LHß, TSHß, POMC, D2 dopamine receptor, histone 3.3 (H3.3), and mouse L-19 ribosomal protein; 25 cycles). 2) At the selected cycle number, different amounts of total RNA were added to ensure that the amplifications of the specific PCR products were linear with respect to input RNA. Mouse L-19 ribosomal protein (L-19) or H3.3 (21) was used as internal control for the normalization of RNA addition for each pituitary mRNA. The PCR products (394 bp GH, 346 bp PRL, 272 bp mouse LHß, 291 bp mouse POMC, 377 bp mouse TSHß, 486 bp D2 dopamine receptor, and 194 bp L-19) were separated on an 8% nondenaturing acrylamide gel, and the PCR products were quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Negative controls included the omission of RNA or reverse transcriptase. Each PCR product was confirmed by dideoxy chain termination sequencing (Sequenase, version 2.0, U.S. Biochemical Corp., Cleveland, OH) after ligation into the pGem-T vector (Promega Corp., Madison, WI).


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  		Table 1. Oligonucleotide primers used for RT-PCR

 
Anterior pituitary cell preparation
Anterior pituitary cell dispersions from 5- to 6-month-old male hGHRH transgenic and control animals were used for the static incubation and cell immunoblot assay. Pituitary cells were prepared as described previously (6). Briefly, after removal of the neurointermediate lobe, the anterior pituitaries were cut into small pieces and incubated with 0.2% trypsin (Worthington Biochemical Corp., Freehold, NJ) at 37 C. After treatment with DNase I (Worthington Biochemical Corp.) and lima bean trypsin inhibitor (Worthington Biochemical Corp.), the cells were dispersed by gentle trituration with a siliconized Pasteur pipette. The cells were counted with a hemocytometer and used for the cell immunoblot assay or static incubation as described below.

Pituitary cell culture: static incubation
Monodispersed anterior pituitary cells from normal (n = 3) and hGHRH transgenic (n = 3) mice were cultured in 96-well tissue culture plates (30,000 cells/well; Nunclon, Thomas Scientific, Swedesboro, NJ) coated with poly-D-lysine (0.5 mg/ml; Sigma). The cells were cultured in phenol red-free DMEM containing 10% gelded horse serum and antibiotics and maintained in a humidified atmosphere of 5% CO2-95% air at 37 C. After 24 h in culture, the cells were washed three times (15 min each time) with serum-free medium 199 (Life Technologies, Inc.) containing 0.1% BSA. The cells were then incubated for 1 h in medium 199-BSA containing 0.1 mM ascorbate or medium 199-BSA containing varying concentrations of dopamine (25, 100, or 400 nM) in 0.1 mM ascorbate. After incubation, the medium was collected and stored at -20 C until assayed for PRL content by RIA as described previously (17).

Cell immunoblot assay (CIBA)/immunocytochemistry
The CIBA procedure (22) relies on the protein adsorbing properties of the Immobilon polyvinylidene difluoride transfer membrane (Millipore Corp., Bedford, MA), and it was performed as described previously (6). Briefly, 1000 cells in 0.2 ml medium were pipetted onto a pretreated piece of Immobilon membrane. The cells were incubated in a water-saturated atmosphere of 5% CO2-95% air at 37 C. After the specified number of hours of incubation, secretion was terminated by fixing the membranes with phosphate-buffered 4% paraformaldehyde for 15 min. The membranes were stored in 0.1 M Sorenson’s buffer at 4 C until immunohistochemical techniques were performed. After incubation and fixation, a 0.05 M Tris-0.05% Tween-20 buffer was applied to the membranes for 15 min. After washing in buffer, 10% normal goat serum was applied to the membranes to block nonspecific binding of antibody. After 1 h, rat anti-PRL (AFP2532690, 1:15,000) in 10% normal goat serum was applied to the membranes, and incubation was continued overnight. The membranes were washed in buffer (three times, 5 min each time), and a biotinylated IgG solution (goat antirabbit; Vector Laboratories, Inc., Burlingame, CA) was applied for 1 h. After another wash in buffer, an avidin-conjugated horseradish peroxidase solution was applied for 1 h, and then a hydrogen peroxide-activated diaminobenzidene chromagen solution (0.5 mg/ml; Sigma) was applied for 4 min. The membranes were washed in buffer, air-dried, and analyzed for hormone secretion as described below. Negative controls included omission of primary antibodies for PRL and omission of secondary antibody. Due to the large degree of variability in immunostaining noted between CIBA experiments, all Immobilon membranes within a given experiment were immunostained on the same day using the same solutions. Secretion areas were analyzed using a Macintosh IIci (Apple Computer, Inc., Cupertino, CA) and NIH Image Analysis software. Fifty cell secretion areas were measured for each animal and averaged to determine one mean hormone secretion area per animal.

Validation of the CIBA
To validate the ability of the CIBA to measure PRL secretion, a series of endocrine manipulations was performed on pituitary cells from hGHRH transgenic mice to determine whether the expected physiological responses could be elicited. To evaluate the effect of estradiol on PRL secretion, three hGHRH transgenic mice were anesthetized with ether, and 2-mm SILASTIC brand capsules (id, 0.020 in.; od, 0.037 in.; Dow Corning Corp., Midland, WI) filled with crystalline 17ß-estradiol (~6 mg crystalline powder) were implanted sc for 1 week. Anterior pituitary cells of the hGHRH transgenic mice that received 17ß-estradiol capsules and three control hGHRH transgenic mice without estradiol treatment were prepared, as described above, for the CIBA. During the in vitro incubation of the pituitary cell preparations from estradiol-treated and control hGHRH transgenic mice, some of the cells were treated with dopamine (500 nM in 0.1 mM ascorbic acid) added to the medium. After each treatment the membranes were immunostained for PRL as described above, and Image Analysis measurements for hormone secretion areas were compared between treatment groups.

TH gene expression
A ribonuclease protection assay was performed to quantify steady state TH mRNA levels in the hypothalamus (RPA II kit, Ambion, Inc., Austin, TX). Total RNA was isolated from hypothalamic fragments that were bordered by the postchiasmatic recess, the premammillary recess, and the parasagittal planes 0.5 mm on either side of the median eminence and were resected to a depth of 2.5 mm. A mouse complementary DNA (cDNA) fragment of TH was obtained by RT-PCR using total RNA isolated from mouse hypothalamus. TH oligonucleotide primers (sense, 5'-GCT GAA GGG CCT CTA TGC TAC TAC CCA TGC CTG-3'; antisense, 5'-GAG TGC AGG AGC TCT CCA TAG GAA GAC AGC-3'; Integrated DNA Technologies, Inc., Coralville, IA) were used for PCR. The single 492-bp PCR fragment was subcloned into pGEM-T (Promega Corp.) and verified by dideoxy chain termination sequencing (Sequenase, version 2.0, U.S. Biochemical Corp.). The TH cDNA template was linearized with SacII and transcribed with SP6 RNA polymerase to produce a 32P-labeled complementary RNA (cRNA) probe. A ß-actin cRNA probe was generated as previously described (21). Twenty micrograms of total RNA were then hybridized with the cRNA probes overnight at 45 C, followed by ribonuclease A/T1 digestion. The protected fragments were separated on a 6% denaturing polyacrylamide gel. The gel was then dried, and the protected bands were quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The ratios of the integrated densities from the TH and ß-actin bands were used for statistical analysis.

Dual in situ hybridization
Dopamine D2 receptor mRNA was localized in lactotrophs and somatotrophs by using dual in situ hybridization. Dr. Daniel I. Linzer (Northwestern University) generously provided the mouse PRL and GH cDNAs. A 486 bp mouse dopamine D2 receptor cDNA was generated in our laboratory by RT-PCR using total RNA from mouse anterior pituitary gland. This cDNA (+1056 to +1541; GenBank accession number X55674) corresponds to a portion of the C-terminal coding region of the mouse dopamine D2 receptor mRNA. The cDNA template was linearized with PstI and transcribed with T7 RNA polymerase in the presence of digoxigenin-UTP to obtain an antisense dopamine D2 receptor cRNA. Dual in situ hybridization was performed on dispersed anterior pituitary cells as described previously (21). The numbers of lactotrophs (cells expressing PRL mRNA) coexpressing dopamine D2 receptor mRNA were counted on slides probed with digoxigenin-labeled dopamine D2 receptor and 35S-labeled PRL cRNAs. The numbers of somatotrophs (cells expressing GH mRNA) coexpressing dopamine D2 receptor mRNA were counted on slides probed with digoxigenin-labeled dopamine D2 receptor and 35S-labeled GH cRNAs. Cells were counted from at least 10 different representative areas (totaling ~500 cells) on each slide, and the mean number of total counts was used to represent the animal (2 slides/animal). Data are presented as the mean ± SEM (n = 3) of the percentage of specific cell types in the anterior pituitary cell population.

Data analysis
All data are expressed as the mean ± SEM. Statistical analyses were performed by one- or two-way ANOVA, followed by Neuman-Keuls multiple range test where appropriate (23). Differences were considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pituitary hormone gene expression
The relative levels of pituitary hormone mRNAs in hGHRH transgenic and control mice (n = 5/experimental group) are shown in Fig. 1Go. Each bar indicates the pituitary hormone mRNA/L-19 ratio that is used to represent the pituitary hormone concentration (pituitary hormone mRNA per µg total RNA in arbitrary OD units). The total RNA content of the anterior pituitary gland of 6-month-old male hGHRH transgenic mice is more than 3-fold greater than that in control mice (2.40 ± 0.23 vs. 0.63 ± 0.07 µg total RNA; P < 0.05). Significant differences were observed in the mRNA concentrations of GH (P = 0.0096), LHß (P = 0.0353), and POMC (P = 0.019); only GH mRNA levels were increased in transgenic mice. When expressed per whole anterior pituitary gland, the mRNAs for GH, PRL, and TSHß were significantly greater (P < 0.05) in hGHRH transgenic mice.



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  		Figure 1. Relative differences in pituitary hormone mRNA levels in the anterior pituitaries of hGHRH transgenic and nontransgenic mice as determined by RT-PCR. Each value represents the mean ± SEM of the pituitary hormone mRNA/L-19 ratio from five animals per group. *, Significantly greater (P < 0.05) than age-matched controls.

 
Pituitary hormone concentrations within the anterior pituitaries of hGHRH transgenic and control mice
Figure 2Go illustrates the protein concentrations of GH, PRL, TSHß, and LHß within the anterior pituitaries of hGHRH transgenic and nontransgenic mice. Each value represents the mean ± SEM of the amount of hormone per mg of total protein (n = 3–11 animals/hormone determination). The total protein content of the anterior pituitary gland of 6-month old male hGHRH transgenic mice is nearly 4-fold greater than that in control mice (18). The concentrations of GH (P = 0.016), TSHß (P = 0.002), LHß (P = 0.0003), and PRL (P = 0.01) were all significantly lower within the hGHRH transgenic mice pituitaries compared with control mice. However, there were no significant differences in GH, TSHß, LHß, or PRL contents when expressed as the amount of hormone per pituitary gland.



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  		Figure 2. Pituitary concentrations of GH (A), PRL (B), TSH (C), and LH (D) in the anterior pituitary glands of hGHRH transgenic and control mice. Hormone contents were determined by RIA and are expressed as amount of hormone per mg protein. Each value represents the mean ± SEM of 3–11 animals/group. *, Significantly greater (P < 0.05) than age-matched controls.

 
Pituitary hormone concentrations in plasma of hGHRH transgenic and control mice
Figure 3Go shows the concentrations of GH, PRL, TSHß, and LHß within the plasma of hGHRH transgenic and nontransgenic mice. The values represent the mean ± SEM of the amount of hormone per ml plasma (n = 3–11 animals/hormone determination). The plasma concentrations of TSHß (P = 0.049) and LHß (P = 0.0004) were significantly lower in the hGHRH transgenic mice compared with nontransgenic animals. The plasma concentrations of PRL were not significantly different (P = 0.1682). In contrast to the anterior pituitary gland content, the plasma concentrations of GH were significantly (P < 0.0001) greater (18-fold) within the hGHRH transgenic mice compared with controls.



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  		Figure 3. Plasma concentrations of GH (A), PRL (B), TSH (C), and LH (D) in hGHRH transgenic and control mice. Hormone levels were determined by RIA and are expressed as amount of hormone per ml plasma. Each value represents the mean ± SEM of 3–11 animals/group. *, Significantly greater (P < 0.05) than age-matched controls.

 
Analysis of PRL secretion from individual mouse anterior pituitary cells and validation of the CIBA
Figure 4Go shows the time-course evaluation of PRL secretion from individual lactotrophs using the CIBA. Two-way ANOVA revealed significant main effects (P < 0.05) of both experimental group and time. Lactotrophs from hGHRH transgenic mice secreted significantly more PRL compared with control animals.



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  		Figure 4. Time-course evaluation of PRL secretion. Average PRL secretion areas from individual anterior pituitary cells of hGHRH and nontransgenic control mice as measured by the CIBA after various periods of incubation. Each value represents the mean ± SEM of 50 PRL-secreting cells averaged per animal (n = 4 animals/group). *, Significantly greater (P < 0.05) than PRL secretion areas from age-matched controls.

 
Endocrine manipulations of PRL secretion produced the anticipated results when using the CIBA. Figure 5Go shows that 1 week of in vivo treatment with estradiol caused a significant increase in the mean secretion areas of PRL (1.4-fold; P = 0.0186) compared with untreated hGHRH transgenic mice. Furthermore, treatment of estradiol-exposed hGHRH transgenic pituitary cells with 500 nM dopamine in vitro significantly decreased the secretion areas of PRL (63% of E2 alone; P = 0.0186) to areas that were not significantly different than PRL secretion from control hGHRH transgenic pituitary cells.



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  		Figure 5. PRL secretion areas from individual hGHRH pituitary cells after in vivo estradiol treatment and in vitro dopamine treatment. Average PRL secretion areas from hGHRH transgenic pituitary cells with or without 1 week in vivo exposure to estradiol (E2). E2-exposed cells were also treated with 500 nM dopamine (DA) in vitro. Each value represents the mean ± SEM of 50 PRL-secreting cells averaged per animal, 3 animals/group, after 2-h incubation. *, Significantly greater (P < 0.05) than control. **, Significantly less (P < 0.05) than E2-treated cells.

 
TH activity in the SME
TH activities in nontransgenic and hGHRH transgenic mice were determined by the accumulation of DOPA in the SME 40 min after NSD-1015 injection. To correct for any differences in tissue size due to dissection of the SME, the data are expressed as nanograms of DOPA per mg total protein. Figure 6Go shows that DOPA accumulation within the SME of the hGHRH transgenic mice was significantly greater (P < 0.05) compared with that in nontransgenic siblings. The average amount of DOPA accumulation in the hGHRH transgenic mouse was 2-fold greater than that found in control mice.



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  		Figure 6. DOPA accumulation in the median eminence of hGHRH transgenic and control mice. DOPA accumulation (nanograms per mg protein) within the SME of hGHRH transgenic and control mice was measured 40 min after treatment with NSD-1015. Each value represents the mean ± SEM of 10 transgenic and 11 control mice. *, Significantly greater (P < 0.05) than age-matched controls.

 
Hypothalamic TH gene expression
Ribonuclease protection assay for TH mRNA resulted in a single protected band of the expected size (data not shown). No significant differences were observed in the hypothalamic content of TH mRNA between hGHRH transgenic and normal mice (Fig. 7Go). Moreover, no differences were observed in TH mRNA concentrations (i.e. TH mRNA per µg total RNA).



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  		Figure 7. TH gene expression in the hypothalamus of hGHRH transgenic and normal mice. TH mRNA levels were quantified by ribonuclease protection assay and are expressed as TH mRNA per µg total RNA. TH mRNA levels were not significantly (P > 0.05) increased in the hypothalamus of hGHRH transgenic mice. Each value represents the mean ± SEM of four or five animals.

 
Pituitary D2 dopamine receptor gene expression: analysis by RT-PCR and dual in situ hybridization
RT-PCR analysis of D2 dopamine receptor gene expression in the anterior pituitary gland resulted in a single PCR product of the expected size (Fig. 8AGo). As shown in Fig. 8BGo, the relative level of D2 dopamine receptor gene expression (per µg total RNA) was 2.5-fold higher (P < 0.05) in hGHRH transgenic mice compared with nontransgenic siblings. If expressed per anterior pituitary gland, D2 dopamine receptor mRNA levels were increased nearly 10-fold in transgenic mice.



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  		Figure 8. Dopamine D2 receptor (D2R) gene expression in the anterior pituitary gland (AP) of hGHRH transgenic and normal (control) mice. A, Representative autoradiogram of RT-PCR in the AP. The photomicrograph shows the relative D2R and histone 3.3 (H3.3) mRNA levels in control (lanes 1–4) and hGHRH transgenic (lanes 5–8) mice. B, Relative differences in D2R mRNA levels were quantified by RT-PCR and are expressed as arbitrary optical density units per µg total RNA. Each value represents the mean ± SEM of four or five animals. *, Significantly greater (P < 0.05) than steady state D2R mRNA concentrations in age-matched controls.

 
Dual in situ hybridization analysis of D2 dopamine receptor gene expression showed that all lactotrophs and greater than 99% of somatotrophs express the D2 dopamine receptor gene in normal and hGHRH transgenic mice (Fig. 9AGo). Moreover, although not quantifiable, the intensity of the immunostaining for the digoxigenin-labeled dopamine D2 receptor cRNA probe was much more intense in hGHRH transgenic mice compared with normal siblings. Nearly 75% of all pituitary cells express the dopamine D2 receptor gene in nontransgenic mice (Fig. 9BGo), and this percentage is significantly (P < 0.05) decreased in hGHRH transgenic mice. The percentage of lactotrophs expressing dopamine D2 receptor mRNA is decreased 50% (P < 0.003) in hGHRH transgenic mice. In contrast, as shown in Fig. 9BGo, the percentage of somatotrophs expressing dopamine D2 receptor mRNA is increased 55% (P < 0.03).



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  		Figure 9. A, Representative photomicrograph of colocalization of dopamine D2 receptor (D2R) and PRL mRNAs in dispersed anterior pituitary cells using dual in situ hybridization. The large arrows point to cells coexpressing D2R and PRL mRNAs, the small arrows point to cells expressing D2R mRNA only, and the arrowheads point to cells expressing neither D2R nor PRL mRNA. B, In situ hybridization analysis of D2R localization in lactotrophs (PRL mRNA-containing cells) and somatotrophs (GH mRNA-containing cells) in normal (control) and hGHRH transgenic mice. Data are expressed as the percentage of total anterior pituitary (AP) cells. Each value represents the mean ± SEM of three animals. *, Significantly different (P < 0.05) from age-matched controls.

 
Lactotroph responsiveness to DA
Exposure of pituitary cells to varying concentrations of DA in vitro showed a differential secretory responsiveness of lactotrophs from nontransgenic vs. hGHRH transgenic mice (Fig. 10Go). PRL secretion during the 1-h static incubation was not significantly different in nontransgenic (5.13 ± 0.38 ng PRL/30,000 pituitary cells) and hGHRH transgenic (5.54 ± 0.37 ng PRL/30,000 pituitary cells) mice. However, we showed previously that although the total number of pituitary cells is increased more than 3-fold, the actual percentage of lactotrophs is decreased nearly 50% in hGHRH transgenic mice (6). Thus, similar to the results obtained by CIBA, the amount of PRL secreted per cell during 1 h of static incubation is greater in lactotrophs from hGHRH transgenic mice. ANOVA showed significant main effects of the concentration of dopamine (P < 0.001) and animal genotype (P < 0.001) on PRL secretion; dopamine inhibited PRL secretion in a concentration- and genotype-dependent manner. Moreover, there was a significant interaction (P < 0.002) indicating that DA inhibited PRL secretion from lactotrophs of hGHRH transgenic mice to a greater extent than it did from lactotrophs of control mice (Fig. 10Go).



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  		Figure 10. Effect of dopamine on PRL secretion from pituitary cells of normal (control) and hGHRH transgenic mice in vitro. Pituitary cells (30,000 cells/well) were exposed to dopamine (25, 100, or 400 nM) or vehicle (0.1 mM ascorbate) for 1 h. PRL levels in the cell culture medium were quantified by RIA. PRL values (mean ± SEM; n = 3/experimental group) are expressed as a a percentage of basal (0.1 mM ascorbate alone) PRL secretion.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ectopic expression of hypothalamic hormones has been implicated in the formation of pituitary adenomas in humans (24, 25, 26). Extrapituitary GHRH-secreting adenomas lead to chronic exposure to excess GHRH resulting in pituitary tumors and the development of acromegaly (25). A similar situation was mimicked by the production of the hGHRH transgenic mouse (2). The hGHRH transgenic mouse displays some of the characteristics of human acromegaly, such as the presence of pituitary adenomas and abnormal increases of body weight and size (2). In the current studies we used the hGHRH transgenic mouse as a model of pituitary tumor formation to characterize the status of pituitary hormones before the emergence of focal pituitary adenomas, with a focus on lactotroph function.

Analysis of mRNA levels for each of the classical pituitary hormones within the anterior pituitaries of hGHRH transgenic and nontransgenic mice revealed that the decreases we observed in the concentrations of pituitary hormone mRNA levels are probably the result of dilution. The anterior pituitaries of the hGHRH transgenic mice are dramatically larger and contain a significantly greater number of cells (6), and therefore, they contain a greater amount of total RNA. Because somatotrophs represent a highly disproportionate amount of the increase in cell types, mRNAs of the other pituitary hormones are much lower in concentration. The actual total absolute amounts of each pituitary hormone mRNA analyzed are greater within the anterior pituitaries of the hGHRH transgenic mice when the data are analyzed as amount of mRNA per pituitary gland. The increases we observed in total GH, LHß, and POMC mRNA levels are proportionate to the increases we reported recently in the numbers of each of their respective cell phenotypes (6). In contrast, the increases in total PRL and TSHß mRNA levels are substantially higher than the increases observed in the numbers of lactotrophs and thyrotrophs.

Interestingly, GH, PRL, and TSHß are the classical pituitary hormones observed to increase within the adenomas that form in the pituitaries of older hGHRH transgenic mice (3). One of the common factors regulating the expression of the GH, PRL, and TSHß mRNAs is the pituitary-specific transcription factor Pit-1 (27). Pit-1 protein expression is essential for the development and maintenance of somatotrophs, lactotrophs, and thyrotrophs (28, 29). Moreover, GHRH stimulates the expression of Pit-1 (30), and Pit-1 immunoreactivity is increased within the hyperplastic anterior pituitaries of hGHRH transgenic mice (31). Thus, it is conceivable that Pit-1 is playing an important role in the aberrant expression of GH, PRL, and TSHß in the hGHRH transgenic mouse.

Further analyses of the hormonal status of the male hGHRH transgenic mouse revealed a decrease in the concentrations of GH, PRL, TSH, and LH within the anterior pituitaries of transgenic mice compared with nontransgenic siblings. Again, the dramatic and nearly selective hyperplasia of somatotrophs is probably causing a dilutional effect on the concentrations of PRL, TSH, and LH. This phenomenon is evident when the data are expressed as the amount of hormone per pituitary. The total amount of each of the hormones is the same within the anterior pituitaries of the hGHRH transgenic and nontransgenic mice. It is puzzling that the amount of GH would be the same in the anterior pituitaries of transgenic and normal mice even though there is over a 4-fold increase in the number of somatotrophs in hGHRH transgenic mice (6). However, the chronic exposure of somatotrophs to elevated levels of GHRH in the transgenic mice leads to continual secretion of GH from somatotrophs as evidenced by the 20-fold increase in the plasma concentrations of GH. The continual release of GH suggests that a reduced amount of GH would remain within the somatotrophs of the transgenic mice.

The plasma concentrations of LH were significantly lowered in the male hGHRH transgenic mouse as well as in some patients who suffer from acromegaly (32). Lowered plasma LH may be the result of elevated levels of SS. SS mRNA levels are elevated in the hypothalamus of the hGHRH mouse (7), and an analog to SS, SMS 201–995, has been shown to decrease the plasma levels of LH in human male volunteers (33). Regardless, the lowered plasma concentrations of LH in male hGHRH transgenic mice do not significantly affect their reproductive functioning as they are used as breeders in our colony of mice.

Plasma TSH concentrations were also significantly lowered in the male hGHRH transgenic mouse. As with LH, plasma TSH levels are lower in acromegalic patients (34, 35). Elevated circulating levels of insulin-like growth factor I (IGF-I) may contribute to the lowered plasma levels of TSH. Recombinant IGF-I has been shown to lower plasma TSH concentrations in healthy male volunteers (36, 37). A similar phenomenon may be present in hGHRH transgenic mice, as they are known to have elevated levels of IGF-I (38).

A common characteristic of acromegalic patients who have tumors that contain mammosomatotrophs is elevated plasma levels of PRL (39, 40). We know from this and one of our previous studies (6) that the anterior pituitaries of hGHRH transgenic mice contain a 2-fold increase in the number of immunoreactive PRL cells as well as a 5-fold increase in the total pituitary content of PRL mRNA. However, there was no concomitant increase in plasma levels of PRL in hGHRH transgenic mice.

Analysis of PRL secretion at the level of the single cell revealed that in vitro removed from hypothalamic influences, lactotrophs from hGHRH transgenic mice secrete greater amounts of PRL than those lactotrophs from nontransgenic mice. Furthermore, absolute values of PRL secretion in a static incubation system confirmed the results of the analysis at the level of the single cell. These results suggest that lactotrophs from hGHRH transgenic mice have the capacity to produce, secrete, and possibly store greater amounts of PRL than lactotrophs from of nontransgenic mice. Yet, plasma levels and total pituitary amounts of PRL in hGHRH transgenic mice are not elevated compared with levels observed in their nontransgenic siblings.

The primary candidate for the in vivo inhibition of PRL production and secretion is hypothalamic dopamine (13). Previous studies using transgenic mice expressing bovine or human GH showed endocrine-dependent alterations in PRL secretion and hypothalamic dopamine turnover rates (41, 42, 43, 44, 45). To assess the dopaminergic tone in male hGHRH transgenic and nontransgenic mice, we performed mRNA and activity analyses of TH, the rate-limiting enzyme in dopamine synthesis (46). We found no significant difference in the steady state levels of TH mRNA within the hypothalamic fragments of the hGHRH transgenic mice. However, our analysis of DOPA accumulation after NSD-1015 administration showed that the rate of synthesis of dopamine is significantly elevated within the hypothalamus of the hGHRH transgenic mice. These data suggest that posttranscriptional mechanisms, such as phosphorylation, are increasing the activity of TH within the tuberoinfundibular neurons in hGHRH transgenic mice, thereby increasing the secretion of dopamine to the anterior pituitaries of these animals.

Another mechanism by which dopamine could exert a greater influence upon the lactotrophs in the hGHRH transgenic mice is through increased sensitivity to dopamine signaling. To evaluate this possibility, first we performed mRNA analyses of the primary pituitary dopamine receptor subtype, D2. We found that the concentrations of D2 dopamine receptor mRNA and overall levels of D2 mRNA were significantly increased within the anterior pituitaries of the hGHRH transgenic mice compared with controls. This finding suggested that cells within the pituitary of the hGHRH transgenic mice may indeed be more sensitive to the levels of dopamine within their extracellular environment. Using varying concentrations of dopamine in vitro, we found that lactotrophs from hGHRH transgenic mice were indeed more sensitive with regard to the inhibition of PRL secretion.

Recent studies of dopamine transporter (47) and dopamine D2 receptor (48) knockout mice showed that changes in dopamine function affected not only PRL gene expression, but also GH gene expression and GHRH production in the hypothalamus. Alterations in dopamine D2 receptor gene expression in the anterior pituitary gland have also been reported in transgenic mice overproducing heterologous forms of GH (49). The cause and effect relationships between D2 dopamine receptor function, and PRL and GH gene expression/secretion still needs to be elucidated in future studies. However, it is interesting to note that the positive correlation between Pit-1 (31) and D2 dopamine receptor gene expression in hGHRH transgenic mice is similar to that observed in human lactotroph and somatotroph adenomas (50). Furthermore, a polymorphism of the dopamine D2 receptor gene was linked recently to idiopathic short stature in children (51).

In conclusion, male hGHRH transgenic mice show a marked hyperplasia of lactotrophs, and a concomitant increase in total PRL mRNA levels. Although hyperprolactinemia would be expected as a result of the proliferation of lactotrophs, plasma PRL levels remain normal. This suggests that feedback mechanisms are responsible for sustaining normal plasma levels of PRL. Our data suggest that changes in hypothalamic tyrosine hydroxylase activity, and pituitary dopamine receptor gene expression and functional sensitivity are able to counterbalance the increase in lactotroph number. At least during the earlier stages of pituitary cell hyperplasia, it appears that increased synthesis and presumed secretion of hypothalamic dopamine in concert with increased dopamine D2 receptor gene expression in the anterior pituitary may maintain normal PRL secretion. We speculate that PRL may be the signal initiating these changes in the hypothalamus and pituitary gland. As lactotrophs begin to proliferate, PRL secretion may be transiently elevated until the hypothalamus and pituitary gland respond accordingly to regulate PRL secretion. These feedback mechanisms may regulate PRL secretion under normal conditions and during the early stages of lactotroph proliferation. We have yet to uncover whether these feedback mechanisms are capable of maintaining normal PRL secretion as the mammosomatotroph adenoma develops in the hGHRH transgenic mouse.


   Acknowledgments
 
We thank Dr. Albert F. Parlow (National Hormone and Pituitary Program, UCLA-Harbor Medical Center) and the NIDDK for generously supplying us with the immunological reagents used in this study. We also thank Drs. Kelly E. Mayo (Northwestern University) and Lawrence A. Frohman (University of Illinois-Chicago) for providing us with the hGHRH transgenic mouse to initiate our breeding colony, and Dr. Daniel I. Linzer (Northwestern University) for providing the mouse PRL and GH cDNAs.


   Footnotes
 
1 This work was supported by NIH Grants DK-45981 (to J.F.H.), HD-07436 (to J.P.M.), HD-24190 (to J.L.V.), and HD-35332 (to L.A.A) and a grant from the National Science Foundation (DBI-9494220; to M.E.H.). Preliminary results of this study were presented at the 27th Annual Meeting of the Society for Neuroscience, New Orleans, Louisiana, 1997. Back

2 Present address: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 4D08, MSC 4150, Bethesda, Maryland 20892-4150. Back

Received August 18, 1999.


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