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Endocrinology Vol. 138, No. 1 379-388
Copyright © 1997 by The Endocrine Society

ARTICLES

Estrogen Regulation of Peptidylglycine {alpha}-Amidating Monooxygenase Expression in Anterior Pituitary Gland

Rajaâ El Meskini, Christine Delfino, Françoise Boudouresque, Micheline Hery, Charles Oliver and L’Houcine Ouafik

INSERM U297, Institut Federatif de Recherche Jean Roche, Faculté de Médecine Nord, 13916 Marseille, Cedex 20, France

Address all correspondence and requests for reprints to: Dr. L’Houcine Ouafik, INSERM U297, IFR Jean Roche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pituitary is a rich source of peptidylglycine {alpha}-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) and peptidyl-{alpha}-hydroxyglycine {alpha}-amidating lyase catalytic domains necessary for the two-step formation of {alpha}-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 ß-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 ß-estradiol treatment induced an overall decrease of the hybridization signal, as compared with OVX rats. Progesterone treatment did not change PAM mRNA levels both in OVX or OVX + E2 rats. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by RT-PCR, it was found that estrogen status does not affect the distribution of PAM mRNA among its various alternatively spliced forms. In OVX 17 ß-estradiol treated rats, the specific activity of PAM in the anterior pituitary decreased in both soluble and particulate fractions compared with OVX animals. Western blot analysis demonstrated a 105-kDa PAM protein in particulate fractions prepared from OVX and OVX-17 ß-estradiol treated animals. The soluble fraction from OVX animals contained major PAM proteins of 105, 95, 84, 75, and 45 kDa, and 17 ß-estradiol treatment caused a decrease in the prevalence of these proteins. These results indicate that estrogens are involved, either directly or indirectly, in regulating the expression of PAM in several cell types in the anterior pituitary gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ONE OF THE STEPS in the synthesis of many bioactive peptides is the conversion of COOH-terminal glycine-extended peptide into {alpha}-amidated product (1, 2). Peptide {alpha}-amidation is a two-step reaction catalyzed by the bifunctional enzyme, peptidylglycine {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3). The PAM precursor protein encodes both of the enzymatic activities involved in peptide amidation (3, 4). The first enzyme, peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) produces an {alpha}-hydroxylated intermediate in the presence of copper, ascorbate, and molecular oxygen. At physiological pH, the second enzyme, peptidyl-{alpha}-hydroxyglycine {alpha}-amidating lyase (PAL), catalyzes the conversion of peptidyl-{alpha}-hydroxyglycine intermediates into {alpha}-amidated peptide and glyoxylate (5, 6). The two catalytic domains of the bifunctional PAM protein can be separated by endoproteolysis and can act independently (7). Soluble and membrane-associated PAM activities have been identified, and their distribution is tissue specific (8). Complementary DNA (cDNA) clones encoding 110-kDa bifunctional integral membrane protein form of PAM have been isolated from many tissues in several species (2). Tissue-specific alternative splicing of the single copy PAM gene can generate at least seven forms of PAM messenger RNA (mRNA) in the rat (2, 9).

Although the major anterior pituitary hormones such ACTH, TSH, FSH, LH, PRL, and GH are not {alpha}-amidated, PAM levels in the anterior pituitary gland are among the highest in rat tissues (10, 11). Several {alpha}-amidated peptides, including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), galanin, neurotensin (NT), joining peptide, GnRH, TRH, and pyroglutamyl-glutamyl-proline amide have been identified in the rat anterior pituitary gland (12, 13, 14, 15, 16, 17, 18).

Evidence for PAM expression in the rat anterior pituitary gland was provided by immunocytochemistry and in situ hybridization (19). The data demonstrated that the anterior pituitary cells intensely immunostained for PAM represented a subpopulation of the gonadotrophs. Surgical ovariectomy has been shown to produce a significant rise in the pituitary content of SP immunoreactivity (IR), NPY-IR, and NT-IR, whereas VIP-IR showed a fall. These changes were reversed with estrogen replacement or high dose estrogen treatment (20). These data prompted us to investigate the effect of estrogens on PAM expression in the anterior pituitary gland.

Although the effects of estrogen status on many cellular metabolic processes have been well documented (21, 22), little is known about their effects on peptide-processing enzymes. In the present study, we have used several techniques to examine the effects of estrogen status on PAM expression in the anterior pituitary gland. Levels of PAM mRNA were evaluated using electrophoretic blot hybridization analysis, and changes in PAM mRNA forms were investigated using RT-PCR. Tissue levels of PAM activity were measured, and PAM protein forms were examined by Western blot analysis. In situ hybridization studies were conducted to determine whether changes in estrogen status altered PAM mRNA levels in all or only a fraction of the total cell population of the anterior pituitary gland. Our results strongly suggest that estrogen status is involved in the regulation of the PAM expression in this tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Female Sprague-Dawley rats (200–250 g; Dépré, Lyon, France) were maintained on a 14-h light, 10-h dark schedule, with food and water provided ad libitum. At least three animals were used for each stage of the estrous cycle and for each experimental group (ovariectomy with or without estrogen replacement). Vaginal smears were studied daily, and animals that exhibited at least four consecutive 4-day estrous cycle were selected for study. The complete study of the estrous cycle was repeated three times.

For studies on hormone replacement, rats were ovariectomized (OVX), and rested for 1 week. Thereafter, several groups of OVX rats received daily sc (sc) injections of 17 ß-estradiol (4, 20, 50, 100 or 1000 µg diluted in 100 µl of sesame oil; n = 3 for each group) (Merck, Germany) for 1 week. The experiment was repeated three times. To study the effect of progesterone on PAM expression, OVX animals received for 1 week daily sc injections of progesterone (P, 1 mg/rat) (Sigma Chemical Co., St. Louis, MO), alone or in combination with 17 ß-estradiol (4 µg/day) or sesame oil (controls).

At the end of each experiment, the animals were killed by decapitation and trunk blood was collected for serum LH and estradiol measurements. Anterior pituitaries were removed rapidly for determination of PAM activity, preparation of total RNA, or in situ hybridization studies.

LH RIA
Plasma levels of LH were measured in duplicate in a single assay by a double antibody RIA and expressed as ng/ml of NIAMDD rat LH-RP 2. Antirat LH serum CSU 120 was provided by Dr G. D. Niswender (Colorado State University). The sensitivity of the assay was 0.25 ng/ml of plasma. The intra and interassay coefficients of variation were below 10%.

Estradiol RIA
Plasma estradiol levels were measured using a RIA (125I-Estradiol COATRIA, BioMerieux, Lyon, France). The cross-reactivity of the antiestradiol antiserum with corticosterone was less than 0.017%. The limit of sensitivity of the assay was 12 pg/ml of plasma. The intra and interassay coefficients of variation were 6% and 7%, respectively.

RNA isolation and Northern blot analysis
Total RNA was prepared from individual anterior pituitaries using the acid guanidinium isothiocyanate-phenol-chloroform procedure (23). RNA (10 µg) was denatured and then electrophoresed on a 1% agarose formaldehyde denaturing gels. The denatured RNAs were transferred to Hybond-N membrane (Amersham Corp, Les Ulis, Paris) by capillary action in 20 x SSC (3.0 M NaCl, 0.3 M sodium citrate, pH 7.0), cross-linked by UV irradiation and hybridized to [{alpha}-32P]-labeled full length PAM cDNA (3.8 kilobase pairs) (24). Filters were prehybridized, hybridized, and washed as previously described (25). To correct for the actual amount of RNA in each lane, blots were stripped and hybridized to cDNA probes derived from frog ribosomal RNA (25). The autoradiograms were analyzed by measurement of optical density by scanner-densitometer using NIH Image 1.54 software (NIH, Bethesda, MD). The results are expressed as optical density (OD) of PAM mRNA/OD 18S rRNA, with the panels representing the mean ± SEM for each group of rats.

Tissue preparation and amidation assays
Anterior pituitary tissue was homogenized in 20 mM NaTES (N-Tris [hydroxy-methyl] methyl-2-amino-ethane sulfonic acid), pH 7.4, 10 mM mannitol, containing 2 µg/ml leupeptine, 16 µg/ml benzamidine, and 300 µg/ml phenylmethylsulfonyl fluoride using a ground glass homogenizer at 4 C. The homogenates were frozen and thawed three times and separated into soluble and particulate fractions, as previously described (25, 26). The crude particulate fractions were solubilized by resuspension in the same buffer containing 1% Triton X-100; after centrifugation for 60 min at 100,000 x g, the supernatants were used to measure solubilized membrane associated PAM activity. All samples were stored at -70 C until time of assay. Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Interchim, Paris) and BSA as standard.

Amidation assays were performed in duplicate essentially as previously described (11, 25). Unless indicated otherwise, assay tubes contained 25,000 - 35,000 cpm of mono-[125I]-D-Tyr-Val-Gly, 0.5 µM D-Tyr-Val-Gly, 500 µM ascorbate, 10 µM Cu2S04, catalase (100 µg/ml), and 2–6 µg protein in 150 mM NaTES buffer, pH 8.5. Reactions velocities are generally expressed as picomoles of product formed per microgram of protein per hour (specific activity). The variation between duplicate samples was less than 5%. The reaction velocities reported are initial velocities, using a concentration of substrate about 10-fold below the Km of the enzyme for peptide substrate. In general, no more than 10% of the substrate was converted into product in the assay.

Western blot analysis
Samples were prepared for electrophoresis by making them 2% in SDS and 5% in 2-mercaptoethanol and boiling for 5 min. Samples of soluble or particulate fractions were fractionated on slab gels containing 10% acrylamide and 0.25% N,N-methylene-bis-acrylamide using the buffer system of Laemmli (27). Proteins were electrophoretically transferred to Hybond-C membranes (Amersham Corp.) for 1 h at 210 mA and visualized with Ponceau S (Sigma). Molecular weights were estimated by comparison with standard protein (Rainbow markers, Amersham Corp.). Hybond-C strips were blocked in PBS buffer containing 5% of nonfat dry milk and immunostained using a 1:2000 dilution of rabbit antiserum raised against purified bacterially expressed PHM [Ab 1764 to rPAM (37–382)], PAL [Ab 471 to rPAM (463–864)] kindly provided by Dr. B. A. Eipper (Johns Hopkins University, Baltimore, MD). Signals were revealed using an enhanced chemiluminescence kit (ECL Kit, Amersham Corp.).

Combined RT-PCR
Total anterior pituitary RNA (5 µg) from OVX or OVX rats treated with 17 ß-estradiol, was reverse transcribed into cDNA using 1 µg oligo(deoxythymidine) 12–18 (Pharmacia PL, Villejuif, Paris) as primer in a 20-µl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM each of four dNTPs (Pharmacia), 20 U RNAsin (Promega, Lyon, France), and 400 U M-MLV reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) at 37 C for 90 min. The synthetic oligonucleotide primers used in the PCR were all 17 mers. Primers yielding sense cDNA were [all base pair numbers are for rPAM-1 (24)] no. 5 (366–382), no.4 (1359–1375), and no. 9 (3124–3140). Primers yielding antisense cDNA were no. 19 (1455–1439), no. 10 (3188–3172) and no. 21 (3834–3816). PCRs were performed in a 50-µl volume, with 20 mM Tris-HCl (pH 7.4, 25 C), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM each of four dNTPs, 1 µM each primer, cDNA derived from the equivalent of 300 ng total RNA, and 2.5 U Extrapol I DNA polymerase (Eurobio, Les Ulis, Paris), samples were overlayed with one drop of light mineral oil and subjected to 35 cycles in a Biometra Trio-Thermoblock cycler (Biometra). Cycling parameters were as follows: the initial denaturation step was performed at 94 C for 5 min; the repeat cycle consisted of annealing at 52 C for 50 sec, followed by extension at 72 C for 2 min and 30 sec and then denaturation at 94 C for 35 sec. The last extension time was lengthened to 10 min. After thermal cycling, most of the oil was manually removed. Samples were fractionated on agarose gels in 89 mM Tris, 89 mM boric acid, and 2.5 mM EDTA, pH 8.0. After staining with ethidium bromide, the gels were photographed. It should be emphasized that internal standards were not included during reverse transcription of amplification, and the amplified products can be compared only in a qualitative manner.

In situ hybridization
In situ hybridization using 35S-labeled riboprobes was performed as previously described (28, 29). Cryostat sections (10 µm) were mounted onto gelatin-coated slides. Labeled probes were prepared using [{alpha}-35S] UTP (New England Nuclear, Dupont, Paris, France) and T3 or T7 RNA polymerase (Stratagene, Paris) to synthesize sense and antisense RNA transcripts, respectively, from the rPAM-1 cDNA (ZAP 6) cloned into Bluescripts (24). RNA sense and antisense transcripts were prepared by linearizing the plasmid with the appropriate restriction enzyme (XbaI for the antisense and Hind III for the sense strand). Slides were fixed in 4% wt/vol paraformaldehyde and acetylated with 0.5% vol/vol acetic anhydride in 0.9% wt/vol NaCl containing 100 mM triethanolamine (pH 8.0). 35S-labeled riboprobe was added to the hybridization buffer (50% formamide, 600 mM NaCl, 1 x Denhardt’s, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM dithiothreitol, 0.2 µg/µl tRNA, and 10% wt/vol dextran sulphate) to give a final concentration of 1 x 106 cpm/50 µl. Hybridization was carried out overnight at 56 C in moist sealed chambers. Coverslips were removed in 2 x SSC. Slides were incubated in a solution of 2 x SSC (30 min, 37 C) containing 10 µg/ml RNase A (Eurogentec, Belgium). Slides were subsequently rinsed in 2 x SSC at room temperature for 30 min, dehydrated in graded ethanol solutions, air dried, and exposed to x-ray film (Kodak X-OMAT AR) for 3 days. For higher resolution analysis, slides were dipped in Ilford K5 photographic emulsion. The slides were stored for 7 days at room temperature, developed, and lightly counterstained with hematoxylin.

Statistical analysis
All results are expressed as the mean values ± SEM. Statistical analysis was performed by a one-way ANOVA followed by Fisher’s PLSD test (ANOVA, Statview 512, Brain Power Inc., Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes of PAM expression during estrous cycle
We first sought to examine the effect of physiological variations of plasma estrogens on PAM mRNA expression by using intact cycling rats. The regularity of estrous cycle was verified by measurement of circulating LH and estradiol levels (Table 1Go). As expected, mean concentrations of LH were greater (P < 0.0001) in rats at 1600 h during the proestrus period and were not different among the other periods of estrous cycle. Similarly, estradiol levels were highest at 1600 h during proestrus period. Anterior pituitary PAM mRNA levels were assessed by Northern blot analysis. Total RNA prepared from the pituitaries of individual rats was subjected to Northern blot analysis, and PAM mRNA was visualized using a radiolabeled rPAM-1 cDNA capable of detecting all of the known forms of rat PAM mRNA (Fig. 1AGo). Pituitary PAM mRNA size ranged from 3.6–3.8 kilobase pairs in size. The PAM cDNA probe was removed from the blots, and the amount of ribosomal RNA present in each sample was determined by hybridization to a cDNA probe for ribosomal RNA (Fig. 1AGo). The amount of PAM mRNA in each sample was then normalized to the amount of ribosomal RNA (Fig. 1BGo). There was a significant change in anterior pituitary PAM mRNA levels during the different periods in cycling female. Anterior pituitary PAM mRNA levels at proestrus and diestrus were significantly lower than at metestrus and estrus. For example PAM mRNA levels at proestrus (1000 h) were 3.0 ± 0.5-fold lower than at metestrus (n = 4; mean ± SEM).


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  		Table 1. LH and estradiol levels in cycling female rats across the estrous cycle

 


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  		Figure 1. Expression of PAM mRNA at different period of estrous cycle in anterior pituitary. A, Aliquot of total RNA (10 µg) from pituitaries of individual rat at specific stage of estrous cycle was fractionated on a denaturing 1% agarose gel and transferred to Hybond-N membrane. The blot was hybridized with a full-length rPAM-1 cDNA probe and exposed to x-ray film at -70 C with an intensifying screen. The blot was subsequently stripped and reprobed with a cDNA probe corresponding to 18S rRNA to permit correction for the amount of sample actually transferred to Hybond-N membrane. B, for densitometric analysis of PAM mRNA levels during estrous cycle, the amount of PAM mRNA were normalized to the amount of ribosomal RNA; this arbitrary ratio was used to express relative tissue PAM mRNA levels. Error bars indicate the SEM. The asterisk indicates that the values for diestrus and proestrus are significantly different from metestrus and estrus values (*, P < 0.05). Similar data were obtained in two additional independent experiments.

 
This suggests that changes in PAM mRNA expression levels in normal cycling females may be related to the changes of estrogen status during the estrous cycle.

Effect of castration and 17 ß-Estradiol replacement (dose-response curve) on PAM expression
To examine the above mentioned hypothesis, adult female rats were OVX and the effect of 17 ß-estradiol replacement on PAM expression was evaluated. Total RNA was prepared from the pituitaries of individual animals and analyzed as described above (Fig. 2AGo). The amount of PAM mRNA in each sample was then normalized to the amount of ribosomal RNA. The data demonstrate that E2 treatment resulted in a 4.5 ± 0.5-fold (n = 3; mean ± SEM) decrease in the level of PAM mRNA in the anterior pituitary gland compared with OVX rats (Fig. 2BGo). Treatment with increasing doses of 17 ß-estradiol reduced PAM mRNA levels to a proestrus value. However treatment with very high doses of 17 ß-estradiol failed to reduce PAM mRNA levels below the proestrus value (Fig. 2BGo). As expected, when OVX animals were treated with increasing doses of 17 ß-estradiol, plasma LH levels declined from high levels to levels below those found in estrus animals (Fig. 2BGo). Based on these results, the OVX animals were treated with 4 µg of 17 ß-estradiol for the subsequent experiments.



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  		Figure 2. Effect of 17 ß-estradiol replacement in OVX rats on anterior pituitary PAM mRNA expression. Total RNA (10 µg) from individual animals in each treatment group was subjected to Northern blot analysis. The blot was hybridized first with the PAM cDNA probe and then stripped and hybridized with the ribosomal RNA probe (A). Quantitative analysis of the blots shown in A was performed as described in Fig. 1BGo (B). Each bar represents the mean ± SEM of three independent experiments. Plasma LH levels in individual (n = 9) animals were measured as described in Materials and Methods.

 
Regulation of PAM expression by estrogen status
In this experimental paradigm, adult rats were OVX; 7 days later one-half of this group received daily sc injection of 17 ß-estradiol (4 µg in 100 µl sesame oil) for 1 week, while the rest received vehicle alone. The efficiency of surgical castration and 17 ß-estradiol treatment was verified for each experiment and resulted in significant alterations in plasma LH concentrations (3.41 ± 0.28 ng/ml vs. 1.55 ± 0.15 ng/ml respectively; P < 0.0001). Anterior pituitary PAM expression in OVX and OVX-17 ß-estradiol treated rats (OVX + E2) was assessed by Northern blot analysis to examine PAM mRNA levels and forms. Total RNA was isolated from the pituitaries of individual rats and PAM mRNA was examined by Northern blot analysis. The results demonstrated a 4.8 ± 0.5-fold decrease in PAM mRNA levels in the OVX + E2 rats as compared with OVX rats (n = 4; mean ± SEM) (data not shown) in agreement with the data shown on Figs. 1Go and 2Go. No alteration in the size distribution of PAM mRNA could be detected.

The effect of estrogen replacement on the anterior pituitary PAM specific activity was also assessed (Fig. 3Go). In OVX rats, total specific PAM activity increased by approximately 2 ± 0.08-fold and was reduced (0.21 ± 0.09 pmol/h) by treatment with 17 ß-estradiol (4 µg/day for 7 days) to levels observed at proestrus period (0.16 ± 0.0015 pmol/h). The majority of the PAM activity (70–80%) was recovered in the soluble fraction independently of estrogen status. In both groups, the specific activity of the soluble and particulate fractions mirrored the total specific activity (Fig. 3Go).



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  		Figure 3. Effect of estrogen status on PAM specific activity in anterior pituitary. PAM specific activity in crude soluble () and particulate (squlo) fractions prepared from three pooled anterior pituitary glands was measured as described in Materials and Methods. Total PAM specific activity (squlf) was calculated by taking into account the amount of protein in the two fractions. Data from three experiments (n = 3 for each treatment group in three experiments) were used to calculate the mean specific activity; each sample was assayed in duplicate. Data are presented as mean ± SEM. The asterisk indicates that the value is significantly different between both groups (*, P < 0.02).

 
The PAM proteins in any tissue reflect both the forms of PAM mRNA present and the co- or posttranslational modifications that occur. To determine whether the forms of PAM protein present in the anterior pituitary (30) were affected by estrogen status, equal amounts of protein (50 µg) prepared from the soluble and particulate fractions from OVX and OVX + E2 rats, were subjected to Western blot analysis (Fig. 4Go). Antibody specific to the PHM (Ab 1764) domain of the rat PAM-1 protein was used to identify PAM proteins present in anterior pituitary extracts (Fig. 4Go). The major PAM protein in particulate fractions from both groups of rats had a mass of 105 ± 3 kDa (Fig. 4AGo) and was also visualized by antiserum (Ab 471) to the PAL domain of the PAM precursor protein (data not shown). The 105-kDa PAM protein found in anterior pituitary membranes is thought to represent rPAM-2 and rPAM-3b. In preparations from OVX + E2 rats, two proteins at 59 kDa and 56 kDa were also present. They were not visualized with a PAL antiserum and their relationship to PAM is unclear. Analysis of the soluble fraction revealed the presence of multiple forms of PAM protein (Fig. 4BGo). Consistent with the effect of estrogen status on PAM specific activity, the soluble proteins from OVX animals yielded a more intense signal than the same amount of protein from OVX + E2 animals. The 105 ± 3 kDa and 45 ± 3 kDa found in the soluble fraction, must be generated by endoproteolytic cleavage between the PAL domain and the putative trans-membrane domain; and between the PHM and PAL domains respectively. Both proteins are products of rPAM-1, whereas the 84 ± 2 kDa PAM protein is a product of PAM mRNA lacking exon A (primarly rPAM-2) (30, 31). The 95 ± 3 kDa PAM protein is thought to represent intact rPAM-3/3a. The 75 ± 2 kDa PAM protein must arise from precursor forms of PAM (rPAM-1, -2, -3a, -3b, or 3) by endoproteolytic cleavage. A 60-kDa protein is also visualized by antibody to PHM, and its relationship to PAM is unclear. The expression of the described PAM proteins was barely detectable in OVX + E2 animals.



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  		Figure 4. Comparison of PAM proteins by Western blot analysis. Aliquots of particulate (A) and soluble (B) fractions prepared from the OVX or OVX + E2 animals were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels. Each of the samples shown in A and B contained 50 µg of protein. Following transfer to Hybond-C membrane, PAM proteins were visualized with antiserum to PHM domain (37–382) and enhanced chemiluminescence kit (ECL Kit). The location of molecular mass markers analyzed in a separate lane are indicated on the left; apparent molecular mass of the various PAM proteins are indicated on the right of A and B. Similar data were obtained in three additional independent experiments.

 
Effect of ovariectomy and progesterone replacement on PAM expression
Adult rats were OVX and the effect of 7-day treatment with progesterone (1 mg/animal) alone and in combination with 17 ß-estradiol (4 µg/animal) was investigated. Total RNA was isolated from the pituitaries of individual rats and PAM mRNA was examined by Northern blot analysis as described above (data not shown). Quantification of the autoradiograms showed that daily administration of progesterone to OVX rats neither affected the ovariectomy induced rise in anterior pituitary PAM transcripts nor interfered with estrogen-induced decrease of PAM mRNA concentrations (Fig. 5Go).



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  		Figure 5. Effect of sex steroids treatment on anterior pituitary PAM mRNA content in OVX rats. After ovariectomy, animals received for 1 week daily sc injection of the vehicle (OVX) or progesterone (P, 1 mg/animal), alone or in combination with 17 ß-estradiol (E2, 4 µg/animal). Total RNA was prepared from each group (n = 4) and analyzed as described in Materials and Methods. Results shown are the mean ± SEM of three independent experiments. The data shows that E2 treatment sharply decreased PAM mRNA levels, whereas treatment with progesterone failed to reverse castration-induced PAM mRNA rise. The asterisk indicates that the value for OVX + E2 and OVX + E2 + P rats is significantly different from OVX and OVX + P rats (*, P < 0.0001).

 
Effect of 17 ß-estradiol treatment on expression of different alternatively spliced forms of PAM mRNA
In the rat, alternative splicing of a single copy PAM gene generates multiple forms of PAM mRNA (Fig. 6AGo) (9, 30, 32). Forms of PAM mRNA lacking all or part of optional exon B [also referred as exon 25 and 26 (9)] are more prevalent in the pituitary tissue (30). The forms of PAM mRNA in the pituitary are poorly resolved on denaturing agarose gels. To determine whether estrogen status altered the forms of PAM mRNA present in the anterior pituitary, cDNA was synthesized by reverse transcription of RNA prepared from OVX or OVX + E2 animals. A qualitative measure of the forms of PAM mRNA present was obtained by carrying out PCR amplification using pairs of oligonucleotide primers that distinguish among the different alternatively spliced forms of PAM mRNA (Fig. 6BGo). The primer pair (5/19) spans the region common to all PAM mRNA forms from the 5' end of rPAM-1 to immediately before optional exon A; the second primer pair (4/10) spanning the regions referred to as exon A and B distinguishes between mRNAs of the rPAM-1 or -2, -3a, -3b, and -3 types; the third pair (9/21) extends from the 3' end of optional exon B to immediately before the putative poly (A) addition signal. The results do not provide a quantitative comparison of levels of PAM mRNA in different samples. Amplified fragments were fractionated on agarose gels (Fig. 6CGo). Based on amplification using these three sets of primers, we found no effect of estrogen status on the alternatively spliced forms of PAM mRNA in the anterior pituitary; this observation is consistent with the results of Northern blot analysis, which failed to demonstrate an alteration in the molecular weight pattern of PAM mRNA after manipulation of estrogen status. Adult anterior pituitary contains only small amounts of rPAM-4 and -5 (30, 32). rPAM-4 and rPAM-5 mRNAs were barely detectable by reverse transcription followed by PCR amplification with form-specific primers and the results demonstrated that estrogen status had no effect on these two forms (data not shown).



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  		Figure 6. PCR analysis of forms of PAM mRNA in anterior pituitary of OVX and OVX + E2 rats. A, Schematic representation of seven forms of rat PAM mRNA (2, 30, 32). Total RNA was extracted and 5 µg was subjected to reverse transcription and PCR amplification as described under Materials and Methods.B, Schematic diagram of rPAM-1 cDNA shows the positions and orientations of oligonucleotide primers used for amplification. The initiation (AUG) and termination (STOP) codons as well as the positions of exon 16 (optional exon A) and exons 25 and 26 (optional exon B) are shown. C, Amplified products were fractionated on 1% agarose gels and stained with ethidium bromide. The number of base pairs present in the amplified products derived from appropriate plasmid controls is indicated on the right; the forms of PAM to which these bands correspond are indicated on the left. The data presented are representative of three individual experiments.

 
In situ hybridization studies
To determine whether alterations in estrogen status affect PAM mRNA expression in a subset of the pituitary cells population, frozen anterior pituitary tissue from OVX or OVX + E2 rats were prepared for in situ hybridization studies. The riboprobe used in this study corresponds to the full-length rat PAM cDNA and was selected for its ability to visualize all of the known forms of PAM mRNA (24, 29). As shown in Fig. 7Go, in OVX rats PAM mRNA was detected in majority of the cell bodies of the anterior pituitary gland. E2 treatment of OVX rats induced an overall decrease of the in situ hybridization signal, suggesting that E2-induced PAM regulation is not restricted to a single cell type.



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  		Figure 7. In situ hybridization for anterior pituitary PAM mRNA in OVX or OVX + E2 rats. Cryostat anterior pituitary sections (10 µm) were hybridized to 35S-labeled antisense PAM riboprobe. Cryosections were dipped in photographic emulsion for cellular resolutions of autoradiographic grains. After a 1-week exposure period, the tissues were processed and stained with hematoxylin. Anterior pituitary glands from OVX (A) or OVX + E2 (B) rats were photographed in the brightfield mode. Both sections showed hybridization of the PAM probe, although less intensely in the section of OVX + E2 rats. Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results demonstrate that PAM expression in the rat anterior pituitary gland is regulated by estrogen status. Indeed, the changes in PAM mRNA levels observed in this study appear to be inversely related to the physiological variations in plasma estrogen levels occurring during the estrous cycle. Furthermore, PAM expression in the anterior pituitary was reduced when OVX rats were chronically treated with estradiol. This negative regulation of PAM gene expression was not restricted to a single subpopulation of cells of the anterior pituitary, as evidenced by our in situ hybridization study. The 4.5 ± 0.5-fold decrease in PAM mRNA levels observed after estrogen treatment of OVX rats resulted in only a 2 ± 0.08-fold decrease in total PAM specific activity. The amount of PAM activity in pituitary extracts represents a balance among synthesis, storage, inactivation, and secretion of the enzyme. Therefore, the changes in PAM mRNA and specific activity may not be quantitatively equals. Similarly, elevated levels of preprohormone mRNA can be accompanied by reduced tissue levels of product peptide if secretion is stimulated sufficiently. All these data indicate that PAM synthesis in the pituitary shows a negative relationship with estrogen status, suggesting that pituitary PAM is regulated mainly by estrogen or an estrogen dependent factor. The molecular mechanism by which steroids modulate the expression of PAM mRNA is not clear. Estrogen receptors are present at high concentrations in the anterior pituitary gland supporting a direct action of estrogens on this tissue (33). Estradiol action on PAM mRNA levels and activity appears to be specific because progesterone has no effect on both parameters.

As indicated in the introduction, several amidated peptides have been identified in the anterior pituitary gland and may act locally in a paracrine/autocrine way (34). Interestingly, the changes in the levels of several {alpha}-amidated peptides in the anterior pituitary gland have been observed after chronic manipulation of estrogen status. Indeed, ovariectomy produced a significant rise in the pituitary content of SP-IR, NPY-IR, and NT-IR. This pattern is reversed with estrogen replacement or high dose estrogen treatment. These changes in peptide content are accompanied by parallel changes in the mRNA levels for each peptide (20). The effect of ovariectomy on anterior pituitary VIP levels are strikingly opposite to those described for SP, NT, and NPY (20). The most dramatic effects were observed following treatment with high dose estrogens, with a large increase in VIP and a significant decrease in NT and SP concentrations. Like VIP, galanin levels increase in the anterior pituitary gland of OVX female rats (15, 35). So far, the simultaneous location of PAM and an amidated peptide in an identified pituitary cell has not been possible. Immunochemical staining of serial pituitary tissue sections for PAM and well-established pituitary hormones has indicated that the anterior pituitary cells intensively stained for PAM represent a subpopulation of the gonadotrophs. PAM has also been identified at moderate levels in corticotrophs and at lower levels in somatotrophs and lactotrophs (19). The location of the amidated peptides in specific pituitary cell types has not been fully established. SP has been detected in rat gonadotrophs and lactotrophs (36); meanwhile, the attempts to colocalize NT in any of the classical anterior pituitary-hormone-producing cells have failed so far. NPY has been localized in thyrotrophs (14) and VIP immunoreactivity has been demonstrated only in lactotrophs (37). Galanin is possibly produced in a number of different pituitary cells, but certainly in lactotrophs (38). Receptors for SP (39), VIP (40), and NT (41) have been identified in anterior pituitary cells. However, there is no clear pattern of their physiological implications in the regulation of pituitary functions. Because their expression has been shown to vary during changes in estrogens levels, the influence of amidated peptides on the pituitary control of reproduction has been investigated. There is now some evidence for a physiological role of NPY in stimulating LH secretion; the association between LH stimulation and increased pituitary NPY expression after ovariectomy further supports a functional role for this peptide in the control of gonadotrophins secretion (42). In contrast, it has been suggested that SP inhibits LH release and LH-induced GnRH release on the basis of passive immunization anti-SP (43). VIP and galanin expression have been mainly associated with PRL secretion. Estrogens stimulate VIP and PRL expression and in vitro studies using dispersed rat lactotrophs support an autocrine role for VIP in the control of PRL secretion (44). Galanin may play a similar role because immunoneutralization of this peptide decreases PRL release (45). The demonstration of PAM regulation in the anterior pituitary gland as well as the production of {alpha}-amidated peptides by this tissue adds further support for the intercellular communications within the anterior pituitary. Indeed, so far, the physiological role of pituitary PAM appears to be limited to the synthesis of amidated peptides which act locally through autocrine and paracrine mechanisms of action (34). Thus, the regulation of PAM activity may represent a third mechanism of feedback of estrogens on pituitary function in addition to those already established at the levels of the hypothalamus and of the pituitary gonadotrophs. This concept may also be extrapolated to thyroid hormones (29).

There is a coordinate regulation of PAM, NPY, SP, and NT expression as already described for PAM and amidated peptides like TRH in developing rat pancreas (46) and POMC (47) in the anterior pituitary under glucocorticoid changes. We have shown that PAM expression is regulated in rat anterior pituitary gland by thyroid hormone status and that pituitary levels of amidated peptides vary in the same direction (29). The opposite changes in PAM and VIP expression in the anterior pituitary gland do not exclude a coregulation of PAM and VIP expression at an individual cellular level. Indeed, a putative coregulation may be blunted by the low percentage of VIP-expressing cells in whole pituitary gland and the analysis of PAM expression in VIP-producing pituitary cells might be necessary for its demonstration.

In summary, the results reported in this study demonstrate that estrogen status regulate PAM expression, a key processing enzyme in the anterior pituitary gland. Our current efforts are directed toward the determination of PAM colocalization with classical pituitary hormones and {alpha}-amidated peptides and the analysis of molecular mechanisms of its regulation by estrogens. Both efforts should help in the understanding of the physiological significance of these hormonal alterations in PAM expression in the anterior pituitary gland.


   Acknowledgments
 
The authors thank Ms. M. Seguin and R. Querat for their excellent secretarial assistance, and F. Youssouf, J. C. Orsoni and H. Gerard for their technical help. We are grateful to G. Anglade for the illustrations and Dr. M. Grino for his critical reading of the manuscript. Reagents for LH RIA were provided by the NIDDK Hormone Distribution Program. The authors thank Dr. Somma-Delpers for determination of plasma estradiol levels. We wish to thank Dr. B. A. Eipper (Johns Hopkins University, School of Medicine, Baltimore, MD) for providing us the antiserum for PAM.

Received July 8, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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