EBM Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blake, C. A.
Right arrow Articles by Helmke, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blake, C. A.
Right arrow Articles by Helmke, S. M.
Experimental Biology and Medicine 230:800-807 (2005)
© 2005 Society for Experimental Biology and Medicine

SYMPOSIA

Estrogen Regulation of the Rat Anterior Pituitary Gland Proteome

Charles A. Blake*,1, Lewis M. Brown{dagger}, Mark W. Duncan{dagger},{ddagger}, Stephen W. Hunsucker{dagger} and Steve M. Helmke{ddagger}

* Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208; {dagger} Department of Pediatrics, Section of Pulmonary Medicine, and {ddagger} Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045

1To whom requests for reprints should be addressed at Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208. E-mail: blake{at}med.sc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and Discussion
 References
 
Estrogen is known to affect the regulation of all six of the established anterior pituitary gland (AP) hormones, but little is known of the specifics of its regulation of the AP hormones, their isoforms, and nonhormonal AP proteins. We used difference gel electrophoresis in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Two-month-old rats were ovariectomized and used at 6 months of age. They were injected subcutaneously with sesame oil vehicle or 50 µg estradiol valerate in vehicle and studied 48 hrs later, approximately 3 hrs before the time of the anticipated onset of the estrogen-induced surges of gonadotropins in blood. The APs were pooled, and the soluble protein fraction was examined in replicate analyses. After DeCyder software analysis, we identified 26 protein spots that had a 1.5-fold or greater average increase in the experimental group relative to the controls. Nineteen showed a 1.5-fold or greater decrease. Estrogen increased levels of the more acidic isoforms of growth hormone and prolactin and of proteins involved in protein synthesis, folding, and secretion (e.g., eukaryotic translation elongation factor 2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit, and secretogranin II) and of some metabolic enzymes (e.g., arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase, and aldolase A). The majority of the downregulated proteins were involved in RNA or DNA interactions (e.g., five heterogeneous nuclear ribonucleoproteins, DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein), but isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III were also reduced. Our results indicate that estrogen acts in vivo within 48 hrs to modulate levels of a significant number of AP proteins.

Key Words: anterior pituitary gland • DIGE • estradiol • growth hormone • mass spectrometry • prolactin • proteome • rat pituitary


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and Discussion
 References
 
Estrogen is an important regulator of the function of the anterior pituitary gland (AP). Changes in ovarian status accompanying puberty, the estrous or menstrual cycle, pregnancy, lactation, aging, ovariectomy, and disease can all modify secretion of the AP hormones (1–5), at least in part, due to altered estrogen secretion. Estrogen can affect hormone secretion by acting directly on the AP, by altering AP responsiveness to hypothalamic factors, or by mediating its actions at the level of the brain where it can alter the secretion of the hypothalamic factors themselves. Pituitary expression of estrogen receptors (ER) and ER mRNA are also modulated by estrogen (6–8), and the ERs have been localized to different AP cell types (9). Estrogen also regulates pituitary mRNA for progesterone receptors (10). It is often difficult to differentiate between these modes of action (3).

Not only does estrogen affect AP hormone synthesis and release, but it can also influence the proportions of hormone isoforms. Postpubertal gonadal regulation of growth hormone (GH) secretion appears to be largely due to estrogens in females and aromatization of androgens to estrogens in males (11–13). Estrogens appear to regulate pulsatile secretion of GH by multiple routes, including shifting the proportions of GH isoforms and altering responsiveness to GH-releasing hormone (14–17). Estrogen also enhances proliferation of lactotropes and increases prolactin (PRL) secretion (18–23). In addition, PRL variants differ with gender and glycosylated PRL is increased by estradiol (24, 25). Estrogen is essential for the regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and release (3, 26–28). Expression of gonadotropin subunits is suppressed or enhanced by estrogen depending on the conditions (29–31). The relative abundance of specific LH and FSH isoforms and thus their bioactivities may also be regulated by estrogen (32–35). In rats, estradiol administration decreases the adrenocorticotropic hormone (ACTH) response to immobilization stress (36). In gonadectomized rats, estradiol increases ACTH in the plasma (37), and ovariectomy decreases the ACTH response to corticotropin-releasing factor (38). In vitro secretion of thyroid-stimulating hormone from pituitaries of ovariectomized rats is increased by low-dose estrogen (39). Estrogen also increased pituitary thyrotropin-releasing hormone receptor mRNA stability and the rate of transcription (40). Together, these data implicate estradiol in the regulation of all six of the established AP hormones. However, little is known of the specifics of its action on the AP hormones, their isoforms, and nonhormonal AP proteins.

Advances in protein separation and mass spectrometry (MS) techniques have markedly enabled our ability to study estrogen-induced effects on proteomes. For example, Molero and colleagues (41) employed 2D gel electrophoresis to demonstrate that 24-hr incubation of bovine vascular smooth muscle cells with 17ß-estradiol causes changes in the expression of several proteins involved in smooth muscle cell proliferation (e.g., {alpha}-enolase isoform 1, vimentin, and HSP-60), cell contraction (e.g., vimentin and caldesmon), and oxidative stress (e.g., protein disulfide isomerase). Recent advances in protein separation and MS techniques also have made it possible to identify large numbers of pituitary proteins. Zhao and colleagues (42) employed a comprehensive multiple gel-based strategy to identify proteins in human pituitary gland homogenates. Their unique approach allowed them to identify a total of 1449 proteins, including low-abundance proteins, membrane proteins, and proteins at the extremes of isoelectric point (pI) and molecular weight. In our present study, we have employed difference gel electrophoresis (DIGE), matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-ToF MS), and peptide mass fingerprinting (PMF) to analyze the effect of short-term estrogen treatment on the AP proteome of long-term ovariectomized rats. In this initial investigation, we limit our report to selected proteins in the soluble protein fraction of the gland.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and Discussion
 References
 
Animals and Collection of Tissue.
Two-month-old rats (CD strain) were ovariectomized by Charles River Laboratories, Inc. (Wilmington, MA) and shipped to the University of South Carolina. Rats were housed two per cage in a room with controlled lighting (lights on 0700–1900 hrs daily) and temperature (20°–22°C) and given Teklad Rodent Diet 8604 (Harlan, Madison, WI) and tap water ad libitum. They were handled three times per week for cage cleaning and used at 16 weeks after ovariectomy at 6 months of age. Animals were maintained and used in accordance with the standards of the Institutional Animal Care and Use Committee of the University of South Carolina.

Control rats (349–430 g) were injected subcutaneously with a total of 0.1 ml sesame oil vehicle (Sigma Chemical Company, St. Louis, MO) into the right and left inguinal region. Experimental rats (361–430 g) were injected with 50 µg estradiol valerate in vehicle. Rats were injected ip approximately 48 hrs later (1100 hrs) with sodium pentobarbital (75 mg/kg body weight) and then were perfused with PBS as described previously (43). The AP of each rat was collected (43), rinsed in PBS, blotted, weighed, placed in a tube embedded in dry ice, and stored at –80°C. The uterine horns in all vehicle-treated animals were small and threadlike; those in all estrogen-treated rats were markedly swollen, indicating that they had been stimulated by estrogen. Careful gross examination of the area of fatty tissue near the end of the uterine horns revealed no evidence of ovarian tissue.

Preparation of Soluble Protein Fraction.
The APs were divided into two pools. Four glands from the vehicle-treated group formed pool A (64.1 mg), and three glands from the estrogen-treated group formed group B (44.0 mg). The APs in a pool were thawed and combined in a 1-ml tissue grinder (Duall 20, catalog no. 885450-0020; Kontes Glass Co., Vineland, NJ). The tissue was homogenized by hand on ice in 10 volumes of buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% TX-100 [Surfact-AmpsX-100; Pierce Biotechnology, Inc., Rockford, IL]) in the presence of protease inhibitor cocktail added at twice the recommended concentration (Complete Mini; Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitor cocktail 1 added at twice the recommended concentration (contains cantharidin, bromotetramisole, and microcystin LR; Sigma). The TX-100 (0.1%) was added to permeabilize any compartments not broken by homogenization and to release soluble components. After homogenization, the sample was treated to digest nucleic acids that interfere with 2D gel resolution (5 mM MgCl2, 200 U/ml DNase I [Sigma; catalog no. D 4513], and 10 U/ml RNase A [Sigma; catalog no. R 5500] for 10 mins on ice). The homogenate was then centrifuged (40,000 g, 30 mins, 4°C). The supernatant containing the soluble protein fraction was collected for protein precipitation as described in the following. Membrane and cytoskeleton/chromatin fractions prepared from the pellet were saved for future analysis.

The protein in the soluble fraction was quantitatively precipitated with methanol and chloroform by the method of Wessel and Flugge (44). Briefly, four volumes of aqueous sample were mixed with four volumes of methanol and one volume of chloroform, vortexed vigorously, and centrifuged. The aqueous phase containing salts and nucleotides was discarded. The protein interface and lower chloroform phase were mixed with three volumes of methanol and centrifuged. The top phase containing lipids was discarded, and the protein pellet was dried in a vacuum centrifuge. The dry protein pellet was resolubilized in reaction buffer (7 M urea, 2 M thiourea, and 4% w/v CHAPS) overnight at room temperature (RT). The dissolved protein was treated with 10 mM DL-dithiothreitol (DTT) to reduce cysteines and to increase protein solubilization. The sample was centrifuged (16,000 g, 10 mins, RT) to remove any undissolved material, mainly small amounts of connective tissue. An aliquot of the solubilized protein was diluted 50-fold and assayed for protein concentration by the method of Bradford (Protein Assay, Bio-Rad Laboratories, Inc., Hercules, CA), and the sample was then adjusted to 5 mg/ml protein with the reaction buffer.

Protein Labeling and 2D Gel Electrophoresis.
Protein was next subjected to minimal CyDye labeling (45–47; CyDye DIGE Fluors [minimal dyes] for Ettan DIGE, GE Healthcare, Piscataway, NJ). For this reaction a 10-µl aliquot (50 µg of total protein) was removed, and the pH was adjusted by addition of pH 8.5 Tris to a final concentration of 30 mM. (The pH was checked with pH paper to ensure that it was near optimal for the labeling reaction.) The CyDye (200 pmoles in 1 µl anhydrous dimethylformamide) was added to the protein and the reaction allowed to proceed (30 mins, 4°C, in the dark). Under these conditions approximately 1% of the lysine residues of the protein are covalently conjugated to the CyDyes (45, 46). The reaction was then quenched by the addition of 1 µl of 10 mM lysine. For each gel the control sample was labeled with Cy3, and the experimental sample was labeled with Cy5. A pool was prepared from small aliquots of each sample and was labeled with Cy2. This pool was used on all gels as an internal standard to allow for more accurate quantification (47). The samples labeled with Cy3 and Cy5 were mixed with an additional 425 µg of unlabeled protein from each of the two samples to facilitate protein identifications after electrophoresis, and then the Cy2 labeled internal standard (50 µg) was added. The mixture was adjusted to a total volume of 450 µl by adding more reaction buffer (i.e., pI 3–10 Pharmalytes [1% final], bromphenol blue [0.001% final], and hydroxyethyl disulfide [0.1 M final]; Destreak; GE Healthcare). Free cysteines reacted with hydroxyethyl disulfide to become disulfide linked to hydroxyethyl groups. During focusing this protects the cysteines from oxidation, which can cause streaking and artifactual spot production in the basic region of 2D gels.

Replicate gels were run (i.e., gel 1 and gel 2). The protein samples (450 µl, 1000 µg of total protein) were passively rehydrated into 24-cm immobilized pH gradient strips (IPG 3–10 NL, GE Healthcare) for 16 hrs and then focused (IPGphor system, GE Healthcare) for 133,000 V·hr (48). Free cysteines were regenerated by reduction. This involves incubating the strips (20 mins, RT) in equilibration solution (6 M urea, 50 mM Tris pH 8.8, 30% [w/v] glycerol, 2% [w/v] SDS, 0.001% bromophenol blue) containing 0.5% DTT. The cysteines were then alkylated by incubation in equilibration solution containing 4.5% iodoacetamide (20 mins at RT). Second dimension separation was performed at 20 W/gel at 25°C (Ettan DALT12 vertical system, GE Healthcare) using hand-cast 8%–16% gradient gels.

Gel Imaging, Spot Picking, and Protein Identification.
The gels were scanned with a Typhoon 9400 Variable Mode Laser Imager (GE Healthcare) set to 100-µm resolution. DeCyder (Differential In-Gel Analysis [DIA]) software version 5.0 (GE Healthcare) was used for spot detection and relative quantification of proteins based on fluorescence images. DeCyder (Biological Variation Analysis [BVA]) software was used to match gels in the study using the internal standard.

For picking spots, gel 2 was poststained with Deep Purple to visualize the unlabeled protein (ca. 99.9% of the total), which does not precisely comigrate with the CyDye-labeled protein. The spots were then excised with a 2.0-mm picking head (Ettan Spot Picker, GE Healthcare) and transferred to 96-well plates for washing and trypsin digestion (Ettan Digester, GE Healthcare). The spots were washed twice with 25 mM NH4HCO3/50% methanol, once with 75% CH3CN, and once with 100% CH3CN. The 96-well plate was allowed to dry, and trypsin was added to each well (10 µl, 10 µg/ml in 20 mM NH4HCO3). The plate was covered and incubated (16 hrs, RT). Digests were mixed with a matrix ({alpha}-cyano-4-hydroxycinnamic acid in 80% CH3CN, 0.1% TFA) and analyzed by MALDI-ToF MS (Voyager DE-PRO; Applied Biosystems, Foster City, CA). Spectral processing included defining the baseline and noise, selection of the monoisotopic peaks, calculation of the signal-to-noise ratios, and calibration on autolytic trypsin peaks. These tasks were performed by ProTS Data (Efeckta Technologies). The output of this program is a list of all peptide masses, excluding trypsin peaks, with a signal-to-noise ratio of >4. The peak list is then submitted to Mascot (Matrix Science Ltd; version 1.9) and PMF performed by matching the entries against the predicted masses of the tryptic fragments generated in silico for each entry in the nonredundant protein database (NCBI nr, November 21, 2004). A mass accuracy of 50 ppm was required for each matching peptide, no post-translational modifications were permitted, but a single missed cleavage was allowed. Searches were not constrained by pI or molecular weight. A P value less than 0.05 was achieved with a MASCOT score of greater than 76. Minimum requirements for positive protein identification were described previously (48).


   Results and Discussion
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
References
 
The DIGE gels are shown in Figure 1Go. We used standard operating procedures to ensure reproducibility. Resolution was optimized by incorporating (i) nuclease to remove nucleic acids, (ii) a methanol/chloroform extraction method to remove salts and lipids, (iii) hydroxyethyl disulfide to protect cysteines, and (iv) optimal isoelectric focusing and SDS-polyacrylamide gel electrophoresis conditions. To assess analytical variability the labeling was repeated, and a second 2D gel was prepared (Fig. 1Go, right panel). The gels are remarkably consistent, indicating the reproducibility of all the discrete steps involved in our sample analysis by DIGE.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 1. Replicate gels demonstrate method reproducibility. The soluble protein fraction from the control rat AP pool A was labeled with Cy3, and the soluble protein fraction from the AP pool B from estrogen-treated rats was labeled with Cy5. For clarity, the internal standard sample labeled with Cy2 is not shown. The acidic region is to the left and higher molecular weight proteins are to the top. Red = upregulation and green = downregulation in estrogen-treated rats relative to controls. Yellow = proteins not differentially regulated. Somatotropin (growth hormone, GH), prolactin (PRL), enolase 1 (ENO1), and eukaryotic translation elongation factor 2 (EF2) are indicated by arrows.

 
The DeCyder software package determined spot boundaries and spot ratios (experimental/control) on each gel. The use of the Cy2 channel facilitated spot matching between analytical replicate gels (47) and allowed for more accurate quantification (48). We considered a spot to be regulated in response to estrogen if there was a 1.5-fold or greater average change in its experimental/control ratio. Forty-five regulated gel spots were identified by MALDI-ToF MS and PMF. Of these, 26 were upregulated and 19 downregulated. Protein spots, with the greatest fold change, between the control (pool A) and estrogen-treated (pool B) groups are listed in Table 1Go.


View this table:
[in this window]
[in a new window]
  		Table 1. Effects on AP protein content of treatment of ovariectomized rats with estrogen. The fold change in spot intensity (estrogen-treated/control) was determined from the Cy5/Cy3/Cy2 images for both gels shown in Figure 1Go. Only those proteins in the soluble protein fraction that had an average fold change of 1.5 or higher are listed. The nominal molecular weight (mol wt) and nominal isoelectric point (pI) were calculated from the sequence of the protein in the database. The measured mol wt and pI of the protein spots were determined from their position on the 2D gel. Isoforms of the same protein are grouped together and placed in order of increasing acidity. The number of peaks submitted to MASCOT, the number of peptide peaks matched to the protein sequence, and their respective MASCOT scores are indicated. The percentage coverage is the amount of the protein sequence covered by the matched peptides. The proteins that increased with estrogen treatment relative to controls have positive fold changes, and the proteins that decreased with estrogen treatment relative to controls have negative fold changes. The fold changes were remarkably consistent between the two gels, indicating the reproducibility of the DIGE-based proteomic analysis.
 
A major advantage of 2D gel strategy is that multiple isoforms of the same protein can be identified. Several examples are evident (Fig. 1Go), and of special interest are the AP hormone isoforms. The large yellow spot in the lower-right quadrant is somatotropin (growth hormone, GH), and two isoforms of increasing acidity are evident. Acidic isoforms have the same molecular weight as the parent protein but lower pIs and so appear to the left of the parent spot. In this report they are numbered in order of increasing acidity. The two acidic isoforms of GH exhibit increased expression after estrogen treatment (Table 1Go). In humans it is known that the acidic GH isoforms are due to phosphorylation of serine and deamidation of glutamine and asparagine (49, 50). Many of the acidic isoforms seen here likely arise via the same mechanisms, emphasizing the importance of using phosphatase inhibitors during sample preparation. Acetylation of the N-terminus or lysine residues can also produce a net acidification by neutralizing a positive charge. The large yellow spot in the lower-left corner is PRL, and to its left is an acidic isoform that is more abundant following estrogen. The DeCyder software also displays three-dimensional topographical images where the intensity of each pixel is represented by height (i.e., Z-axis, Fig. 2Go). This is an important tool for checking the spot boundaries determined by DeCyder DIA and for checking that the pattern of spots is correctly matched between the gels. Here the control sample (Cy3) is shown on the left and the estrogen-treated sample (Cy5) on the right. The 3D representations can be rotated and viewed from any angle so that differences can be clearly seen and evaluated. In this example the increase in the acidic isoform of PRL is clearly evident, while the surrounding spots are unchanged. The large spot of PRL, partially shown, is unchanged.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. A 3D topographical representation of the PRL region of the 2D gel. The intensity of the CyDye labeled proteins is shown by the height. The control sample (left) and the estrogen-treated sample (right) are similar except for the increase in the level of the PRL acidic isoform.

 
Other proteins important to AP function also show multiple isoforms. The row of spots indicated to the top right is composed of closely related forms of elongation factor 2 (EF2), a protein involved in protein synthesis. The row of spots near the middle of the gel is composed of closely related forms of enolase 1, a protein involved in glycolysis, growth control, and many other functions. Their red color indicates their very strong upregulation by estrogen. Detailed analysis of the isoforms of enolase 1 is shown in Table 1Go. All four spots have the same molecular weight (slightly less than the predicted molecular weight due to removal of the signal sequence), but only one spot has the predicted pI. The more acidic spots are likely due to phosphorylation or possibly deamidation or acetylation. These changes either add a negatively charged phosphate group to neutral residues such as serine, threonine, and tyrosine; convert neutral residues such as glutamine and asparagine to negatively charged glutamic acid and aspartic acid residues; or neutralize a positively charged residue. As the isoforms become progressively more acidic, the number of peptides matched decreases, and therefore the percentage coverage decreases. The disappearance of some peptides from the MALDI-ToF MS spectrum may be due to the fact that acidic modifications make them more difficult to ionize, but because the acidic isoforms are themselves less abundant, specific peptides may simply be below the signal-to-noise threshold. Acidic isoform 3 was present at a very low level, and this alone accounts for the lower number of matched peptides and sequence coverage. Tandem MS experiments are currently under way to identify the precise nature and site of the modifications responsible for the isoforms of enolase, GH, and PRL.

Our data demonstrate that treatment with estrogen produces numerous changes in the soluble AP proteome, including increases in the more acidic isoforms of GH and PRL. These data indicate that rat studies are relevant to humans because these isoforms have previously been described in humans and are known to have important biological consequences. There were also increases in the levels of several proteins involved in protein synthesis, folding, and secretion, such as EF2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit (archain 1), and secretogranin II. The EF2, shown in Figure 1Go, catalyzes the translocation of peptidyl-tRNA from the ribosomal A site to the P site in translational elongation. It has been suggested that the control of the protein translation rate is influenced by phosphorylation/dephosphorylation of EF-2 (51, 52). Several interesting metabolic enzymes were also upregulated, including arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase M1/M2, and aldolase A. Arginosuccinate synthetase is involved in the regulation and detoxification of nitric oxide, an important signaling molecule. Enolase 1 has been implicated in growth control and regulation of c-myc. DRP-2 (TOAD-64, CRMP-2) is activated with cell division. Programmed cell death 6 interacting protein was also upregulated. The majority of the downregulated proteins are involved in RNA or DNA interactions. These include five heterogeneous nuclear ribonucleoproteins (hnRNPs), A1, A2/B1, K, L, and F. Also reduced were DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein. The changes in this group of proteins may reflect large-scale alterations in gene expression at this early time point following estrogen treatment. Other down-regulated proteins were isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III. Downregulation of secretogranin III concurrent with secretogranin II upregulation indicates alterations in the secretory process. Doubtless many other changes will be revealed with analysis of the membrane and cytoskeleton/chromatin fractions.

The results of this study clearly indicate that estrogen acts in vivo to regulate a significant number of proteins and some of their acidic isoforms in the AP-soluble fraction. However, the data do not enable us to determine whether estrogen acted directly on the AP or indirectly via the hypothalamus or by some other means. We also do not know whether changes in content of an individual protein occurred in one or more of the AP cell types. Compared to a rat with intact ovaries, the experimental design was biased toward analyzing gonadotropes. Ovariectomy causes an increase in AP weight (53), primarily because of an increase in number and size of gonadotropes (54, 55). The APs used in the present study weighed approximately 50% more than sham-operated, aged-matched animals whose AP proteome was not analyzed. We employed a protocol for administering estrogen that exerts a biphasic effect on gonadotropin secretion. Initially estrogen exerts a negative feedback on LH and FSH release (56). This is followed by positive feedback as evidenced by surges of LH and FSH in blood (57). The rats in the present study were studied approximately 3 hrs before the anticipated onset of the surges. Thus, estrogen-induced changes in the AP proteome may be associated with effects on gonadotropes related to negative feedback or associated with prepositive feedback, other AP cell types, or all cell types in the glandular epithelium.


   Footnotes
 
This work was supported by a grant from the National Institutes of Health (MD00233).


   References
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 

  1. Baird DT, Scaramuzzi RJ. Changes in the secretion of ovarian steroids and pituitary luteinizing hormone during the periovulatory period in the ewe: the effect of progesterone. J Endocrinol 70:237–245, 1976.[Abstract/Free Full Text]
  2. Karsch FJ, Foster DL, Legan SH, Ryan KD, Peter GK. Control of the preovulatory endocrine events in the ewe: interrelationship of estradiol, progesterone and luteinizing hormone. Endocrinology 105:421–426, 1979.[Medline]
  3. Blake CA. Gonadotropin secretion, control of. In: Knobil E, Neill JD, Eds. Encyclopedia of Reproduction, New York: Academic Press, pp528–552, 1999.
  4. Correa da Costa VM, Moreira DG, Rosenthal D. Thyroid function and aging: gender-related differences. J Endocrinol 171:193–198, 2001.[Abstract]
  5. Kudielka BM, Buske-Kirschbaum A, Hellhammer DH, Kirschbaum C. HPA axis responses to laboratory psychosocial stress in healthy elderly adults, younger adults, and children: impact of age and gender. Psychoneuroendocrinology 29:83–98, 2004.[Medline]
  6. Schreihofer DA, Stoler MH, Shupnik MA. Differential expression and regulation of estrogen receptors (ERs) in rat pituitary and cell lines: estrogen decreases ER{alpha} protein and estrogen responsiveness. Endocrinology 141:2174–2184, 2000.[Abstract/Free Full Text]
  7. Tena-Sempere M, Barriers ML, Gonzalez LC, Pinilla L, Aguilar E. Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary. Neuroendocrinology 74:347–358, 2001.[Medline]
  8. Tena-Sempere M, Navarro VM, Mayen A, Bellido C, Sanchez-Criado JE. Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary. Biol Reprod 70:671–678, 2004.[Abstract/Free Full Text]
  9. Mitchner NA, Garlick C, Ben-Jonathan N. Cellular distribution and gene regulation of estrogen receptors {alpha} and ß in the rat pituitary gland. Endocrinology 139:3976–3983, 1998.[Abstract/Free Full Text]
  10. Szabo M, Kilen SM, Nho SJ, Schwartz NB. Progesterone receptor A and M messenger ribonucleic acid levels in the anterior pituitary of rats are regulated by estrogen. Biol Reprod 62:95–102, 2000.[Abstract/Free Full Text]
  11. Wehrenberg WB, Giustina A. Basic counterpoint: mechanisms and pathways of gonadal steroid modulation of growth hormone secretion. Endocr Rev 13:299–308, 1992.[Medline]
  12. Kerrigan JR, Rogol AD. The impact of gonadal steroid hormone action on growth hormone secretion during childhood and adolescence. Endocr Rev 13:281–298, 1992.[Medline]
  13. Weissberger AJ, Ho KK. Activation of the somatotropic axis by testosterone in adult males: evidence for the role of aromatization. J. Clin Endocr Metab 76:1407–1412, 1993.[Abstract]
  14. Leung KC, Howe C, Gui LY, Trout G, Velduis JD, Ho KK. Physiological and pharmacological regulation of 20-kDa growth hormone. Am J Physiol Endocrinol Metab 283:E836–E843, 2002.[Abstract/Free Full Text]
  15. Kimura N, Tomizawa S, Arai KN, Kimura N. Chronic treatment with estrogen up-regulates expression of sst2 messenger ribonucleic acid (mRNA) but down-regulates expression of sst5 mRNA in rat pituitaries. Endocrinology 139:1573–1580, 1998.[Abstract/Free Full Text]
  16. Veldhuis JD, Evans WS, Bowers CY. Estradiol supplementation enhances submaximal feed-forward drive of growth hormone (GH) secretion by recombinant human GH-releasing hormone-1–44-amide in a putatively somatostatin-withdrawn milieu. Clin Endocr Metab 88: 5484–5489, 2003.
  17. Silverman BL, Kaplan Sl, Grumbach MM, Miller WL. Hormonal regulation of growth hormone secretion and messenger ribonucleic acid accumulation in cultured bovine pituitary cells. Endocrinology 122: 1236–1241, 1988.[Abstract]
  18. Yin P, Kawashima K, Arita J. Direct actions of estradiol on the anterior pituitary gland are required for hypothalamus-dependent lactotrope proliferation and secretory surges of luteinizing hormone but not of prolactin in female rats. Neuroendocrinology 75:392–401, 2002.[Medline]
  19. Christian HC, Morris JF. Rapid actions of 17beta-oestradiol on a subset of lactotrophs in the rat pituitary. J Physiol 539(Pt 2):557–566, 2002.[Abstract/Free Full Text]
  20. Aoki Mdel P, Orgnero E, Aoki A, Maldonado CA. Apoptotic cell death of oestrogen activated lactotrophs induced by tamoxifen. Tissue Cell 35:143–152, 2003.[Medline]
  21. Denef C. Paracrine control of lactotrope proliferation and differentiation. Trends Endocrinol Metab 14:188–195, 2003.[Medline]
  22. Pelletier G, Li S, Phaneuf D, Martel C, Labrie F. Morphological studies of prolactin-secreting cells in estrogen receptor alpha and estrogen receptor beta knockout mice. Neuroendocrinology 77:324–333, 2003.[Medline]
  23. Gomez O, Balsa JA. Autocrine/paracrine action of pituitary vasoactive intestinal peptide on lactotroph hyperplasia induced by estrogen. Endocrinology 144:4403–4409, 2003.[Abstract/Free Full Text]
  24. Sinha YN, Jacobsen BP. Glycosylated PRL in the murine pituitary: detection by a novel assay and alteration of concentrations by physiological and pharmacological stimuli. Biochem Biophys Res Commun 148:505–514, 1987.[Medline]
  25. Anthony PK, Stoltz RA, Pucci ML, Powers CA. The 22K variant of rat prolactin: evidence for identity to prolactin-(1–173), storage in secretory granules, and regulated release. Endocrinology 132:806–814, 1993.[Abstract]
  26. Brann DW, Mahesh VB. Regulation of gonadotropin secretion by steroid hormones. Front Neuroendocrinol 12:165–207, 1991.
  27. Shupnik MA. Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone. Biol Reprod 54:279–286, 1996.[Abstract]
  28. Ulloa-Aguirre A, Maldonado A, Damian-Matsumura P, Timossi C. Endocrine regulation of gonadotropin glycosylation. Arch Med Res 32: 520–532, 2001.[Medline]
  29. Kaynard AH, Low KG, Melner MH. Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones. Mol Cell Endocrinol 88:67–75, 1992.[Medline]
  30. Kawakami W, Winters SJ. Regulation of luteinzing hormone secretion and subunit messenger ribonucleic acid expression by gonadal steroids in perifused pituitary cells from male monkeys and rats. Endocrinology 140:3587–3593, 1999.[Abstract/Free Full Text]
  31. Christin-Maitre S, Laveille C, Collette J, Brion N, Reginster JY. Pharmaco-dynamics of follicle stimulating hormone (FSH) in post-menopausal women during pulsed estrogen therapy: evidence that FSH release and synthesis are controlled by distinct pathways. J Clin Endocr Metab 88:5405–5413, 2003.[Abstract/Free Full Text]
  32. Kojima FN, Cupp AS, Stumpf TT, Zalesky DD, Roberson MS, Werth LA, Wolfe MW, Kittok RJ, Grotjan HE, Kinder JE. Effects of 17ß-estradiol on distribution of pituitary isoforms of luteinizing hormone and follicle-stimulating hormone during the follicular phase of the bovine estrous cycle. Biol Reprod 52:297–304, 1995.[Abstract]
  33. Zambrano E, Olivares A, Mendez JP, Guerrero L, Diaz-Cueto L, Veldhuis JD, Ulloa-Aguirre A. Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution throughout the human menstrual cycle. J Clin Endocrinol Metab 80:1647–1656, 1995.[Abstract/Free Full Text]
  34. Damian-Matsumura P, Zaga V, Maldonado A, Sanchez-Hernandez C, Timosse C, Ulloa-Aguirre A. Oestrogens regulate pituitary alpha 2,3-sialyltransferase messenger ribonucleic acid levels in the female rat. J Mol Endocrinol 23:153–165, 1999.[Abstract]
  35. Moore IG, Ng Chie W, Hudson NL, McNatty KP. Isoforms and half-life of FSH from sheep with different reproductive states. J Endocrinol 165:185–192, 2000.[Abstract]
  36. Young EA, Altemus M, Parkison V, Shastry S. Effects of estrogen antagonists and agonists on the ACTH response to restraint stress in female rats. Neuropsychopharmacology 25:881–891, 1997.
  37. McCormick CM, Linkroum W, Sallinen BJ, Miller NW. Peripheral and central sex steroids have differential effects on the HPA axis of male and female rats. Stress 5:236–247, 2002.
  38. Wilson ME, Mook D, Graves F, Felger J, Bielsky IF, Wallen K. Tamoxifen is an estrogen antagonist on gonadotropin secretion and responsiveness of the hypothalamic-pituitary-adrenal axis in female monkeys. Endocrine 22:305–315, 2003.[Medline]
  39. Moreira RM, Lisboa PC, Curty FH, Pazos-Moura CC. Dose-dependent effects of 17-beta-estradiol on pituitary thyrotropin content and secretion in vitro. Braz J Med Biol Res 30:1129–1134, 1997.[Medline]
  40. Kimura N, Arai K, Sahara Y, Suzuki H, Kimura N. Estradiol transcriptionally and posttranscriptionally up-regulates thyrotropin-releasing hormone receptor messenger ribonucleic acid in the rat pituitary cells. Endocrinology 134:432–440, 1994.[Abstract]
  41. Molero L, Garcia-Mendez A, Alonso Orgaz S, Carrasco C, Macaya C, Lopez Farre AJ. Proteomic approach to identify changes in protein expression modified by 17beta-estradiol in bovine vascular smooth muscle cells. Clin Sci (Lond) in press, 2005.
  42. Zhao Y, Giorgianni F, Desiderio DM, Fang B, Beranova-Giorgianni S. Toward a global analysis of the human pituitary proteome by multiple gel-based technology. Anal Chem 16:5324–5331, 2005.
  43. Blake CA, Helmke SM. Proteomics of the anterior pituitary gland as a model for studying the physiology of a heterogeneous organ. Exp Biol Med (Maywood) 230:793–799, 2005.[Abstract/Free Full Text]
  44. Wessel D, Flugge UI. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143, 1984.[Medline]
  45. Unlu M, Morgan, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077, 1997.[Medline]
  46. Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396, 2001.[Medline]
  47. Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, Currie I. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3:36–44, 2003.[Medline]
  48. Nicolls MR, D’Antonio JM, Hutton JC, Gill RG, Czwornog JL, Duncan MW. Proteomics as a tool for discovery: proteins implicated in Alzheimer’s disease are highly expressed in normal pancreatic islets. J Proteome Res 2:199–205, 2003.[Medline]
  49. Zhan X, Giorgianni F, Desiderio DM. Proteomics analysis of growth hormone isoforms in the human pituitary. Proteomics 5:1228–1241, 2005.[Medline]
  50. Baumann G. Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr Rev 12:424–449, 1991.[Medline]
  51. Yan L, Nairn AC, Palfrey HC, Brady MJ. Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells. J Biol Chem 278: 18177–18183, 2003.[Abstract/Free Full Text]
  52. Sans MD, Xie Q, Williams JA. Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini. Biochem Biophys Res Commun 319:144–151, 2004.[Medline]
  53. Olson DR, Blake CA. Basal luteinizing hormone and follicle-stimulating hormone release rates as a function of time after castration in female and male rats. Neuroendocrinology 53:124–133, 1991.[Medline]
  54. Tougard C, Kerdelhue B, Tixier-Vidal A, Jutisz M. Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique. J Cell Biol 58:503–521, 1973.[Abstract/Free Full Text]
  55. Garner LL, Blake, CA. Ultrastructural, immunocytochemical study of the LH secreting cell of the rat anterior pituitary gland: changes occurring after ovariectomy. Biol Reprod 24:461–474, 1981.[Abstract]
  56. Blake CA. A medial basal hypothalamic site of synergistic action of estrogen and progesterone on the inhibition of pituitary luteinizing hormone release. Endocrinology 101:1130–1134, 1977.[Medline]
  57. Blake CA. Effects of estrogen and progesterone on luteinizing hormone release in ovariectomized rats. Endocrinology 101:1122–1129, 1977.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blake, C. A.
Right arrow Articles by Helmke, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blake, C. A.
Right arrow Articles by Helmke, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS