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Endocrinology, doi:10.1210/en.2003-0646
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Endocrinology Vol. 145, No. 2 604-612
Copyright © 2004 by The Endocrine Society

Functional Modification of Pituitary Somatotropes in the Aromatase Knockout Mouse and the Effect of Estrogen Replacement

Ming Yan, Margaret E. E. Jones, Maria Hernandez, Dongling Liu, Evan R. Simpson and Chen Chen

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Chen Chen, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: chen.chen{at}phimr.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Available data on the influence of estradiol (E2) on GH levels remains controversial. A factor contributing to this uncertainty is a lack of knowledge of both E2 action on somatotropes as well as the molecular mechanisms involved. In this study we investigated gene expression implicated in GH secretion in somatotropes derived from female aromatase knockout (ArKO) mice. In these mice E2 production is blocked due to disruption of the Cyp19 gene encoding aromatase, the enzyme responsible for estrogen biosynthesis. The effect of E2 replacement was also studied by in vivo treatment of mice with E2 for 3 wk. It was demonstrated that somatotropes from ArKO mice had a low expression of GH, GH secretagogue receptor, GHRH receptor (GHRH-R), and pituitary-specific transcription factor (Pit-1). On the other hand, the somatotropes exhibited elevated expression of somatostatin receptors (sst1–5). Overall, these effects resulted in a reduction in GH secretion. E2 replacement increased GHRH-R, Pit-1, and GH mRNA levels to 185%, 193%, and 157% and reduced the levels of sst1, sst2, sst4, and sst5 mRNA expression in ArKO mice, respectively. E2 replacement did not affect the levels of pituitary estrogen ({alpha} and ß) and androgen receptor mRNA expression. It is concluded that the expression of important genes involved in GH synthesis in somatotropes of the female ArKO mouse are functionally down-regulated, and such a down-regulation is reversed to normal levels by E2 replacement. The levels of GH secretagogue receptor, GHRH-R, and Pit-1 mRNA expression were also reduced, and sst1 and sst3 mRNA expression enhanced in aging ArKO and wild-type mice, resulting in a decrease in GH mRNA expression. It is suggested that aging is another important impact factor for the pituitary expression and regulation of GH mRNA in female mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH LEVELS vary during different developmental stages. GH levels increase during puberty, peak in late puberty, and decrease in older age (1). The regulation of GH secretion by estradiol (E2) and aging are still issues that are not well understood (1). Available data about the influence of E2 on GH levels remain controversial. Ample evidence indicates that E2 replacement exerts effects on GH secretion depending upon the route, dosage, and duration of administration. For example, oral E2 administration stimulates GH secretion in girls and normal women (2) and increases GH release in postmenopausal women. Transdermal E2 (100-µg patches applied twice weekly) exerts a lesser effect on GH (3). However, transdermal E2 at higher doses (0.1-mg estraderm patch, two patches changed daily for 2 wk) significantly increases GH secretion with decreases in IGF-I levels in postmenopausal women (4). Short-term (6–23 d) E2 replacement in postmenopausal women enhances the GH-releasing effect of GH releasing peptide-2 (GHRP-2), which is capable of activating an endogenous GHRP receptor pathway (5). Long-term (3 yr or more) oral estradiol therapy in postmenopausal women has a positive effect to increase circulating GH (6). Age plays a significant role in the declining GH levels and GH axis activity of woman (7, 8). For example, older reproductive-aged (42–46 yr) women have decreased GH concentrations compared with younger controls (19–34 yr) (7). Furthermore, GHRH-stimulated GH levels were significantly reduced in postmenopausal woman (45.3–71.8 yr), suggesting that there is a decrease in somatotropic axis activity with aging (8).

Neuroendocrine control of GH secretion is regulated by at least three specific hypothalamic hormones, GHRH, GH secretagogues (GHS), and somatostatin (SRIF). Five different SRIF receptor subtypes (sst1 to sst5) have recently been cloned and characterized in various species, including human, rat, and mouse (9, 10). Activation of GHRH or GHS receptors (GHRH-R and GHS-R, respectively) activates Gs and Gq11 proteins on the somatotrope cell membrane, respectively, resulting in an increase in GH secretion. In contrast, SRIF receptor binding activates an inhibitory Gi protein, which suppresses the signal transduction systems for both GHRH and GHS, leading to a reduction of GH secretion (11, 12). However, how E2 modulates GH secretion through GHRH-R, GHS-R, and SRIF receptors remains unclear. For example, E2 replacement can double the response to a GHRP-2 stimulus in healthy postmenopausal women, implying that E2 may up-regulate GHRP receptor (GHS-R)/effector activity (5) or stimulate ghrelin, an endogenous GHS-R agonist, to regulate GH release (12). Other studies suggest that E2 either suppresses (short-term, 5-d treatment) (13) or does not affect pituitary GHRH-R mRNA expression in female adult oophorectomized rats (14). On the other hand, E2 was reported to have a differential action on distinct pituitary SRIF receptors (sst1–5). For example, it decreased the levels of sst1 and increased sst2 and sst3 mRNA expression in rat pituitary cells (15). In addition, E2 down-regulated the expression of sst5 mRNA in ovariectomized rat pituitary glands (16).

Although E2 is an obvious candidate to increase GH secretion in women, the sites of action of E2 in the GH regulatory axis are still not well understood (17). The E2-deficient aromatase knockout (ArKO) mouse is an ideal model for clarifying this issue and also for checking the reversibility with E2 replacement. ArKO mice lack a functional Cyp19 gene encoding aromatase, the enzyme responsible for the synthesis of androgen to estrogen, and consequently cannot synthesize E2 (18). The expression of GH, GHRH-R, GHS-R, pituitary-specific transcription factor (Pit-1), and sst1–5 genes in pituitary glands from ArKO or control mice with or without E2 replacement was measured in the present experiments. The specific aims were to determine 1) whether E2 deficiency affects pituitary GH secretion; 2) whether E2 deficiency influences the levels of pituitary GH, GHS-R, GHRH-R, Pit-1, and sst1–5 mRNA expression; 3) whether exogenous E2 replacement reverses the changes in somatotropes in ArKO mice; and 4) whether aging and the associated hypoestrogenism resulting from physiological ovarian failure affect the levels of pituitary GH, GHS-R, GHRH-R, Pit-1, and sst1–5 mRNA expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and pituitary tissues
ArKO mice were generated by disrupting the Cyp19 gene by homologous recombination as previously described (18). Heterozygous females were bred to produce wild-type (WT) and homozygous-null offspring. Mice were genotyped by PCR as previously reported (18). Animals were maintained on a soy-free diet as previously reported (19) under specific pathogen-free conditions and had unlimited access to drinking water. Experimental female mice were divided into four groups: 1) WT (n = 6), 2) WT plus E2 replacement (n = 6), 3) ArKO (n = 4), and 4) ArKO plus E2 replacement (n = 4). Female aging mice at 18 months were divided into two groups: 1) WT (n = 6), and 2) ArKO (n = 7). For E2 replacement, female ArKO mice were implanted with 21-d release 17ß-estradiol or placebo pellets [0.05 mg 17ß-estradiol/pellet; 0.002 mg/d; body weight (BW), 24.5 g; 0.097 mg/kg·d; Innovative Research of America, Toledo, OH] at 7–10 wk of age. After 21 d, pituitary tissues were removed, and the wet mass was measured and frozen at -70 C until analyzed. All tissues were stored and analyzed separately.

RIA for serum GH
The concentration of serum GH was measured in duplicate by a double-antibody RIA using kits provided by the National Hormone and Pituitary Program, NIH (mouse GH and mouse GH antisera). The sensitivity of the assay was 0.3 ng/ml, and the inter- and intraassay coefficients of variation were less than 15% and 8%, respectively (n = 6). All samples from one experiment were measured in the same assay, and GH values were expressed as nanogram equivalents of the mouse GH standards supplied by the U.S. National Hormone and Pituitary Program.

RT-PCR
Total RNA was extracted via the Qiagen Ready Kit (Qiagen, Hilden, Germany) from mouse pituitary tissues. RT was performed using the RT system (Promega Corp, Madison, WI). RNAs (1.0 µg) were reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega Corp.) using random hexanucleotide primers. cDNA was amplified by means of PCR using the specific sets of primers in Master PCR buffer (Promega Corp.; Table 1Go). The thermal cycling profiles for GH, GHS-R, GHRH-R, Pit-1, estrogen receptor {alpha} (ER{alpha}), ERß, and androgen receptor (AR) were as follows: 94 C for 5 min, 30 cycles of 94 C for 30 sec at 56 C [GH, GHS-R, GHRH-R, Pit-1, and glyceraldehyde-3-phosphate dehydrogenase {alpha} (GAPDHa)] or 57 C (ER{alpha}, ERß, and AR), 72 C for 45 sec, and 72 C for 10 min. The thermal cycling profiles for SRIF receptors (sst1, sst2, sst3, sst4, and sst5) and GAPDHb were 94 C for 5 min, 35 cycles of 94 C for 1 min, 54 C for 1 min, 72 C for 1 min, and 72 C for 10 min. The PCR products were separated on 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Non-reverse transcribed RNA was included with the primer sets as a negative control, as previously described (20). Amplification of a portion of the GAPDH (housekeeping) gene served as a control for the efficiency of RNA isolation and cDNA synthesis. The GH, Pit-1, GHRH-R, GHS-R, ER{alpha}, ERß, AR, and sst1–5 mRNA levels were normalized to the corresponding GAPDH mRNA level.


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  		TABLE 1. Sequences of primers of RT-PCR for mouse pituitary GH, GHS-R, GHRH-R, Pit-1, sst1-5, GAPDH, ER, and AR mRNA levels

 
Validation of the RT-PCR for pituitary GH, GHS-R, GHRH-R, Pit-1, and sst1–5 mRNA
In preliminary experiments we determined the linear phase of the PCR for each set of primers, and appropriate cycle numbers were chosen to measure the expression of target genes (Fig. 1Go). All transcripts were amplified with similar efficiencies between 25 and 35 cycles for GH, GHS-R, GHRH-R, Pit-1, and GAPDHa (Fig. 1AGo) as well as ER{alpha}, ERß, and AR (data not shown) and between 30 and 45 cycles for sst1–5 and GAPDHb (Fig. 1BGo). Similar results were obtained in at least three independent experiments of at least four mice. The validity of above RT-PCR assays for semiquantitative analysis is supported by 1) the selection, for each target, of amplification conditions in the exponential phase of PCR; 2) amplification efficiencies that are similar among all transcripts within each reaction; 3) the use of an appropriate internal control; and 4) no competition between primer sets (sst1–5 and GAPDHb). Thus, the PCR amplification of GH, GHS-R, GHRH-R, Pit-1, and GAPDHa transcripts was conducted using 30 amplification cycles, and that of sst1–5 and GAPDHb was performed using 35 cycles, respectively.



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  		FIG. 1. Optimization of RT-PCR conditions for semiquantitative analysis of expression of the target mRNAs. For amplification in the exponential phage of PCR, different numbers of cycles were tested for GH, GHS-R, GHRH-R, Pit-1, and sst1–5 messages. All transcripts were amplified with similar efficiencies between 25 and 35 cycles for GH, GHS-R, GHRH-R, Pit-1, and GAPDHa (A) and between 30–45 cycles for sst1–5 and GAPDHb (B). The linear phase of the PCR for each set of primers and appropriate cycle numbers (30 cycles for GH, GHS-R, GHRH-R, Pit-1, and GAPDHa; 35 cycles for sst1–5 and GAPDHb) were chosen to measure the expression of target genes. The signal intensities of the PCR products (upper panels of A and B) were measured by pixel density quantified by image analysis software. All PCR products were of the expected sizes (Table 1Go).

 
Statistical analysis
Results are presented as the mean ± SEM ratio to GAPDH mRNA. Results for same group were obtained in at least three independent experiments of at least four mice. The data for animals in the same diestrous stage were analyzed by two-way ANOVA for main effects of genotype and treatment group and their interaction, followed by Tukey’s post hoc one-way ANOVA. Statistical significance was declared for differences with P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BW, pituitary weight (PW), and pituitary RNA concentration of female adult mice
There were no significant changes in BW, PW, pituitary RNA concentration, or PW/BW ratio between WT and ArKO groups. After E2 replacement, PW, amount of pituitary RNA recovered, and PW/BW ratio were increased, but BW was not significantly changed in WT and ArKO groups (Table 2Go). These results suggested that E2 replacement increases PW and RNA levels in female WT and E2-deficient ArKO mice.


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  		TABLE 2. BW, PW, and pituitary RNA concentration with E2 treatments in female ArKO and WT mice

 
Serum GH concentration and levels of mRNA encoding for GH and GAPDH in female adult mice
After abolishing Cyp19 gene function, the serum GH level decreased significantly to 60% of the WT level (Fig. 2Go), and pituitary GH mRNA expression was significantly decreased (Fig. 3AGo). To confirm the results we compared the levels of GH and GAPDH mRNA expression in four groups, respectively. GAPDH mRNA expression was not significantly different in all experimental groups with E2 or without E2 replacement (Fig. 3BGo). As such, the ratio of GH to GAPDH mRNA expression was significantly reduced in female ArKO mice. E2 replacement markedly increased the level of GH mRNA expression to 157% of that in ArKO mice (Fig. 3CGo).



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  		FIG. 2. GH concentrations in serum of female adult ArKO mice, aged 7–14 wk. *, P < 0.05 compared with control level of GH.

 


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  		FIG. 3. GH and GAPDH mRNA levels in pituitaries of WT and ArKO mouse with or without E2 replacement. A, GH mRNA expression. B, GAPDH mRNA expression. C, Ratio of GH and GAPDH mRNA levels. *, P < 0.05 compared with mRNA levels in WT mice; #, P < 0.05 compared with mRNA levels in ArKO mice.

 
Levels of pituitary GHS-R, GHRH-R, Pit-1, sst1–5, ER, and AR mRNA expression in female adult mice
In female E2-deficient ArKO mice, the levels of mRNA encoding for GHS-R, GHRH-R, and Pit-1 were significantly reduced (Fig. 4Go). E2 replacement increased the levels of GHRH-R and Pit-1 mRNA expression to 185% and 193% of those in ArKO mice, but there was no significant increase in GHS-R (Fig. 4Go). By contrast, none of the treatment significantly affected the levels of pituitary ER ({alpha} and ß) and AR mRNA expression in ArKO mice (Figs. 5Go and 6Go).



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  		FIG. 4. The levels GHS-R, GHRH-R, and Pit-1 mRNA in pituitaries of WT and ArKO mice with or without E2 replacement. *, P < 0.05 compared with mRNA levels in WT mice; #, P < 0.05 compared with mRNA levels in ArKO mice.

 


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  		FIG. 5. The levels of ER-{alpha} and ER-ß mRNA in pituitaries of WT and ArKO mice with or without E2 replacement.

 


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  		FIG. 6. AR mRNA levels in pituitaries of WT and ArKO mice with or without E2 replacement.

 
All five SRIF receptor mRNAs were identified in normal pituitary glands of female adult mice. The expression of each SRIF receptor subtype in descending order was: sst2 = sst5 > sst4 > sst3 > sst1 (Fig. 7Go). The pituitary mRNA levels of all five SRIF receptor subtypes were elevated in ArKO mice (Fig. 7Go). E2 replacement reduced the levels of sst1, sst2, sst4, and sst5 mRNA expression in ArKO mice respectively (Fig. 7Go). These results indicate that sst2, sst4, and sst5 are the major SRIF receptor subtypes expressed in the pituitary glands of female adult mice, and that all three are positively regulated by E2.



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  		FIG. 7. The levels of sst1–5 mRNA in pituitaries of WT and ArKO mice with or without E2 replacement. *, P < 0.05 compared with mRNA levels in WT mice; #, P < 0.05 compared with mRNA levels in ArKO mice.

 
These data suggest that E2 replacement can reverse the decrease in pituitary GH mRNA expression in female E2-deficient ArKO mice. This is associated with up-regulation of levels of GHRH-R and Pit-1 transcripts and down-regulation of sst1, sst2, sst4, and sst5 mRNA expressions.

Levels of pituitary GHS-R, GHRH-R, Pit-1, and sst1–5 mRNA expression in aging (18 month old) female mice
In female aging (18 month old) WT mice, the levels of mRNA encoding pituitary GH, GHS-R, GHRH-R, and Pit-1 were significantly reduced compared with those in young adult WT mice (Fig. 8Go). Although the levels of GH and GHRH-R mRNA expression were greater in the aging ArKO group than in the aging WT group, GH mRNA was expression still lower than that in young adult WT mice (Fig. 8Go).



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  		FIG. 8. The levels of GH, GHS-R, GHRH-R, and Pit-1 mRNA in pituitaries of young adult and aging (18 month old) WT and ArKO mice. *, P < 0.05 compared with young adult WT mice; #, P < 0.05 compared with aging WT mice.

 
All five SRIF receptor mRNAs were expressed in normal pituitary glands of aging female mice (Fig. 9Go). The pituitary levels of sst1 and sst3 mRNA expression were significantly increased in the aging WT and ArKO groups, and sst4 and sst5 were reduced in the aging WT group compared with those in young adult WT mice (Fig. 9Go). These results suggest that the expression of both sst1 and sst3 is positively regulated by aging.



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  		FIG. 9. The levels of sst1–5 mRNA in pituitaries of young adult and aging (18 month old) WT and ArKO mice. *, P < 0.05 compared with the young adult WT mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study is that somatotropes from E2-deficient ArKO mice have a low expression of GHRH-R and GHS-R, and a high expression of SRIF receptors, leading to a reduction in GH mRNA expression and plasma GH concentration. E2 replacement increased pituitary Pit-1 and GHRH-R and reduced sst1, sst2, sst4, and sst5 mRNA expression, resulting in the increase in GH mRNA expression in ArKO mice. Thus, the expression level in somatotropes is returned to normal levels after E2 treatment in ArKO mice. Aging significantly down-regulated the levels of pituitary GHS-R, GHRH-R, and Pit-1 and enhanced sst1 and sst3 mRNA expression, resulting in decreasing GH mRNA expression compared with adult mice, implying that aging is an important impact factor for the pituitary expression and regulation of GH mRNA in female mice.

E2 plays an important role in GH secretion, but the mechanism of the regulation of GH secretion by E2 is not clear (2, 11, 12). Exogenous E2 exerts disparate effects on the somatotropic axis that appear to be dependent on the dosage, route, and duration of administration. For example, transdermal E2 at lower doses (0.7 and 2.0 µg/kg·d) did not have any significant effect on GH and IGF-I levels as assessed by challenge testing (21). However, most E2 replacement studies measuring the effects on the GH axis suggest that hormone replacement can restore and stimulate GH secretion (4, 22, 23). High doses of oral E2 (31 µg/kg·d) or transdermal E2 (3.0 µg/kg·d) significantly increased GH secretion (4). Although E2 is an obvious candidate to regulate GH secretion in women, the sites and mechanisms of action of E2 on the GH axis (hypothalamus, pituitary, or peripheral tissues) remain unclear (17). As E2 treatment at higher doses and long term for 3 yr increased circulating GH and decreased IGF-I in postmenopausal women (4, 6), in the present study estrogen at higher doses (97 µg/kg·d) and long term (3 wk) was used in female ArKO mice.

Previous studies have demonstrated that E2 stimulates GH release via effects on the hypothalamic secretion of SRIF and GHRH (24), and E2 administration to ovariectomized ewes with hypoestrogenism increased GH pulse amplitude (25). The present results indicate that somatotropes in adult ArKO mice are functionally down-regulated by the reduction in expression of important genes involved in GH secretion, and such a down-regulation is reversed back to normal by E2 replacement. However, the mechanism by which E2 regulates GH secretion through GHS-R, GHRH-R, Pit-1, and SRIF receptors remains unclear. GHS-R is a recently cloned specific receptor that may be part of an endocrine pathway controlling GH secretion (26). GHS-R mRNA is expressed not only in the pituitary gland, but also in other tissues, including the brain in both hypothalamic and nonhypothalamic regions (27, 28). GHS-R expression was markedly up-regulated by GHRP and thyroid hormone and down-regulated by GH (11, 26). The current data indicate that GHS-R mRNA expression was reduced in ArKO mice, but was not significantly increased with E2 replacement. The mechanism, however, is not clear, and it may be that E2 enhances GH secretion, and GH feedback down-regulates GHS-R expression in ArKO mice (11, 26). GHRH-R mRNA is mainly expressed in the pituitary gland in a temporal and spatial pattern corresponding to GH gene expression (29). E2 treatment (25 µg/kg·d; daily injection for 5 d) either decreased pituitary GHRH-R or GH mRNA levels (13) or did not affect GHRH-R mRNA expression in the oophorectomized rat (14). In contrast, in E2-treated rat pituitary cells, GHRH increased GH release, and somatostatin lost its inhibitory action on GH secretion (30). The GH response to GHRH significantly decreased in premenopausal women after ovariectomy, implying that hypoestrogenism reduced the role of the GHRH/GHRH-R pathway, and E2 replacement therapy restored GH to presurgical levels, suggesting that E2 increases the GH response to GHRH/GHRH-R in E2 deficiency (31). Our study indicated that in E2-deficient (ArKO) mice, GHRH-R and Pit-1 expression were down-regulated, and E2 replacement reversed GHRH-R and Pit-1 expression to normal levels. This may be because Pit-1 is proposed to directly stimulate transcription of GHRH-R, leading to increased numbers of GHRH-R on the surface of somatotropes (29, 32). Pit-1 regulates the expression of GH in response to changes in the hormone environment, but little is known about the physiological regulation of Pit-1 in the pituitary (33, 34). The present study indicates that Pit-1 plays an important role in the regulation of pituitary GH mRNA expression. Pit-1 mRNA expression was down-regulated and correlates with the decreases in GH, GHS-R, and GHRH-R mRNA levels, and such a down-regulation is reversed back to normal by E2 replacement. Pit-1 is actively regulated physiologically and may be involved in mediating the effects of sex steroids (34). Pit-1 activates transcription of the GH gene, leading to an increase in GH to replenish cellular hormone stores in somatotropes (32, 34).

Five different SRIF receptor subtypes (sst1 to sst5) have recently been cloned and characterized in various species, including human, rat, and mouse (9, 10). The mouse anterior pituitary expresses all five subtypes (35). However, the functional significance of these subtypes has not yet been clarified. To verify whether SRIF receptor gene expression is related to the change in GH mRNA expression, the transcript levels of the five SRIF receptor subtypes were measured in the present study. It was demonstrated that sst2, sst5, and sst4 were the forms predominantly expressed. Our results are in agreement with those of others who reported a similar pattern of expression in human and rat pituitary cells (36, 37, 38). In those reports the expression of each SRIF receptor subtype in descending order was: sst5 > sst2 > sst4 = sst3 > sst1. sst2 and sst5 also appear to be the dominant subtypes in the human somatotrope (36, 37, 38). High levels of sst4 and sst5 receptor mRNA were expressed in rat somatotropes (39). sst2 and sst5 transcripts were more abundant in somatotropes and thyrotropes than in the other cell types in rats (40). Although the expression of each SRIF receptor subtype changes in diseases such as diabetes and pituitary adenomas, little is known about the subtype expression in E2 insufficiency. sst1, sst2, and sst3 mRNA levels are decreased in the fasted rat, and sst1, sst2, sst3, and sst5 mRNA levels are decreased in the streptozotocin-induced diabetic rat (41), both of which are characterized by low circulating GH levels.

In the present report the pituitary mRNA levels of all five SRIF receptor subtypes were elevated in E2 insufficiency (ArKO). With E2 replacement, sst1, sst2, sst4, and sst5 mRNA levels were reduced in ArKO mice. Levels of sst3 transcripts were not altered. E2 is known to have a differential effect on the different pituitary SRIF receptors. For example, 1 µM estradiol treatment decreased the level of sst1 and increased sst2 and sst3 mRNA expression in rat pituitary cells (15). Treatment with 1 mg E2 once a week for 4 wk increased the expression of sst2 mRNA in rat pituitary (42). In contrast, E2 treatment suppressed the inhibitory role of the somatostatin/SRIF receptor pathway on GH release in rat pituitary cells (30) and down-regulated the pituitary expression of sst5 mRNA in ovariectomized rats (42). Our study indicated that E2 decreases sst5 mRNA expression in ArKO mice similar to the result observed in the ovariectomized rat (42). Studies have shown that E2 treatment down-regulates ER-{alpha} and ER-ß mRNA in the hypothalamus (43). However, E2 does not alter the expression of ER-{alpha} or ER-ß in rat pituitary glands (44). Our study also supported the latter results. Such a difference in region-specific regulation of ER may be related to the following findings. 1) Few pituitary GH cells contain ER, and E2 action through the ER{alpha} is likely to be minimal. In addition, few somatotropes are estrogen receptive (45). 2) ER dynamics vary markedly between pituitary tissues and hypothalamus (46). For example, the rate of replenishment of cytosolic ER is slower in pituitary (24 h or more) and faster in hypothalamus (12 h). A transient loss of total ER after E2 injection occurs in pituitary, but is absent in hypothalamus (46).

Pituitary concentrations of GH, GHRH-R, and Pit-1 mRNAs were lower in 1-yr-old rats than in adult (49 d old) rats (47). Our results also indicate that the levels of pituitary GH, GHRH-R, GHS-R, and Pit-1 mRNA expression were significantly reduced in aging (18 months) female WT mice with physiological ovarian failure. Although the levels of GH and GHRH-R mRNA expression were greater in the aging ArKO than in the aging WT group, GH mRNA expression was lower than that in young adult WT mice. The reason for higher levels of expression of GH and GHRH-R mRNAs in aging ArKO mice than aging WT mice is not clear. A possible reason may be the increase in androgen levels in ArKO mice (19).

The patterns of SRIF receptor subtype mRNA expression with aging are still unknown (48, 49). For example, a study in rats showed that sst2 mRNA abundance rose 6-fold at 10 wk of age compared with that in 2-d-old pituitaries, but did not change compared with that in glands from 1-yr-old animals (48). However, another study using rats indicated that the levels of pituitary sst2 and sst5 mRNA declined after 16 months (49). In our study we showed that with aging all five SRIF receptor subtype mRNAs were expressed in pituitary glands of female mice, and that aging positively regulated sst1 and sst3 mRNA expression. In addition, our study indicated that sst2, sst4, and sst5 were decreased in aging (18 month old) female WT mice. We speculate that the different results from different studies are due to different ages and species employed. For example, 12- to 14-month-old rats are middle-aged, and 22- to 24-month-old rats are old (50). In the rat, sst2 and sst5 are the main receptors mediating the suppressive action of SRIF on GH, but in the human, sst5 is critical for physiological regulation of GH (51).

We conclude that the reduction of pituitary GHS-R, GHRH-R, and Pit-1 and the increase in SRIF receptor mRNA expression levels are associated with endogenous E2 deficiency in female mice, implicating all GHS-R, Pit-1/GHRH-R, and SRIF receptor signaling systems as control points in the establishment and maintenance of the effect of endogenous E2 on GH mRNA expression. Conversely, exogenous E2 replacement increases the pituitary level of GH mRNA, corresponding to up-regulation of levels of GHRH-R and Pit-1 and down-regulation of sst1, sst2, sst4, and sst5 mRNA expression. This indicates that the Pit-1/GHRH-R and SRIF receptor signaling systems are the primary mediators of pituitary GH mRNA expression by estrogens. Aging also significantly reduced the levels of GH, GHS-R, GHRH-R, and Pit-1 transcripts and enhanced sst1 and sst3 mRNA expression, consistent with the decrease in GH secretion that is known to occur with age.


   Acknowledgments
 
We thank Drs. G. Y. Nie and G. Ooi for their help with molecular biology methods, and Drs. W. C. Boon and Y. Murata, Ms. E. Gong and F. Brennan, and Mr. Y. Zhao and S. Chu for their help with this study. We also thank Ms. S. Panckridge for graphical preparation, and Dr. A. F. Parlow (National Hormone and Pituitary Program, NIDDK) for the mouse GH RIA kits.


   Footnotes
 
This work was supported by Australian National Health and Medical Research Council and United States Public Health Service Grant R37-AG-08174.

Abbreviations: AR, Androgen receptor; ArKO, aromatase knockout; BW, body weight; E2, estradiol; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHRH-R, GHRH receptor; GHRP-2, GH releasing peptide-2; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; Pit-1, pituitary-specific transcription factor; PW, pituitary weight; SRIF, somatostatin; sst, somatostatin receptor subtype; WT, wild-type.

Received May 27, 2003.

Accepted for publication October 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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