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Endocrinology Vol. 139, No. 10 4092-4101
Copyright © 1998 by The Endocrine Society

ARTICLES

Effects of Castration and Chronic Steroid Treatments on Hypothalamic Gonadotropin-Releasing Hormone Content and Pituitary Gonadotropins in Male Wild-Type and Estrogen Receptor-{alpha} Knockout Mice

Jonathan Lindzey, William C. Wetsel, John F. Couse, Tammy Stoker, Ralph Cooper and Kenneth S. Korach

Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology (J.L., J.F.C., K.S.K.) and Hormone Action Group (W.C.W.), Laboratory of Signal Transduction, National Institute for Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; the Department of Psychiatry and Behavioral Sciences, Duke University (W.C.W.), Durham, North Carolina 27710; the Reproductive Toxicology Division, Environmental Protection Agency (T.S., R.C.), Research Triangle Park, North Carolina 27711

Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Laboratory of Reproductive and Developmental Toxicology, National Institute for Environmental Health Science, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testicular androgens are integral components of the hormonal feedback loops that regulate circulating levels of LH and FSH. The sites of feedback include hypothalamic areas regulating GnRH neurons and pituitary gonadotropes. To better define the roles of androgen receptor (AR), estrogen receptor-{alpha} (ER{alpha}), and estrogen receptor-ß (ERß) in mediating feedback effects of sex steroids on reproductive neuroendocrine function, we have determined the effects of castration and steroid replacement therapy on hypothalamic GnRH content, pituitary LHß and FSHß messenger RNA (mRNA) levels, and serum gonadotropins in male wild-type (WT) and estrogen receptor-{alpha} knockout (ERKO) mice. Hypothalami from intact WT and ERKO males contained similar amounts of GnRH, whereas castration significantly reduced GnRH contents in both genotypes. Replacement therapy with estradiol (E2), testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic GnRH content in castrated (CAST) WT mice; only the androgens were effective in CAST ERKOs.

Analyses of pituitary function revealed that LHß mRNA and serum LH levels in intact ERKOs were 2-fold higher than those in intact WT males. Castration increased levels of LHß mRNA (1.5- to 2-fold) and serum LH (4- to 5-fold) in both genotypes. Both E2 and T treatments significantly suppressed LHß mRNA and serum LH levels in CAST WT males. However, E2 was completely ineffective, and T was only partially effective in suppressing these two indexes in the CAST ERKO males. DHT treatments stimulated a 50% increase in LHß mRNA and serum LH levels in WT males, whereas serum LH was significantly suppressed in DHT-treated ERKO males.

Although the pituitaries from intact ERKO males contained similar amounts of FSHß mRNA, serum FSH levels were 20% higher than those in the intact WT males. Castration increased FSHß mRNA levels only in WT males, but significantly increased serum FSH levels in both genotypes. Both E2 and T treatments significantly suppressed serum FSH in CAST WT males, whereas only E2 suppressed FSHß mRNA. DHT treatments of CAST WT mice stimulated a small increase in serum FSH, but failed to alter FSHß mRNA levels. None of the steroid treatments exerted any significant effect on FSHß mRNA or serum FSH levels in CAST ERKOs.

These data suggest that hypothalamic GnRH contents can be maintained solely through AR signaling pathways. However, normal regulation of gonadotrope function requires aromatization of T and activation of ER{alpha} signaling pathways in the gonadotrope. In addition, serum FSH levels in male ERKOs appear to be regulated largely by nonsteroidal testicular factors such as inhibin. Finally, these data suggest that hypothalamic ERß may not be involved in mediating the negative feedback effects of T on serum LH and FSH in male mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MALE vertebrates, the levels of circulating LH and FSH are largely determined by the stimulatory effects of GnRH and activins (1, 2) and the inhibitory effects of steroids and inhibin (1, 2, 3). The negative feedback effects of testicular androgens on serum LH and FSH were first demonstrated in studies in which castration resulted in substantial increases in circulating LH and FSH that were prevented if physiological levels of testosterone (T) were administered (4). Subsequent studies have demonstrated that the feedback effects of T are mediated at the level of the hypothalamus and the pituitary. Although there are conflicting data concerning the effects of T on GnRH synthesis and secretion, a number of studies have demonstrated that castration and steroid replacement alter levels of GnRH messenger RNA (mRNA) (5, 6, 7), processing of GnRH prohormone (8, 9, 10, 11), hypothalamic GnRH contents (12, 13, 14, 15, 16), and patterns of pulsatile GnRH release (16, 17, 18, 19).

Besides examining hypothalamic sites of action, a number of investigators have also examined feedback directly on the pituitary. T, dihydrotestosterone (DHT), or estradiol (E2) can suppress GnRH-stimulated LH secretion from pituitary cultures (20, 21), whereas T treatments can increase basal FSH secretion and intrapituitary FSH levels (22). Furthermore, molecular analyses of the promoter regions of the gonadotropin genes have revealed that some of the feedback effects of E2 or T may be mediated through androgen receptor (AR) or estrogen receptor (ER) interactions with response elements and other transcription factors that regulate expression of the {alpha}-gonadotropin subunit ({alpha}GSU), and the FSHß and LHß subunits (1, 2, 23).

Although these data support both hypothalamic and pituitary sites of steroid feedback, it is unclear whether the effects of T are primarily mediated directly through the AR or indirectly via aromatization and activation of ER. Support for AR-mediated feedback is found in studies in which treatments with DHT (a nonaromatizable androgen) effectively suppressed serum LH and steady state levels of {alpha}GSU and LHß mRNA in rats (24, 25), androgen blockade with flutamide (an antiandrogen) resulted in elevations of serum LH (26), and AR-mediated suppression of the {alpha}GSU gene expression occurred through enhancer elements in the promoter region of that gene (23). Convincing arguments for the role of AR are also found in models of receptor-based androgen resistance, the testicular feminized mouse (Tfm) (27) and human androgen insensitivity syndromes (28), in which serum LH can be significantly elevated. By contrast, other studies have demonstrated robust suppression of serum LH and FSH, and gonadotropin mRNAs by exogenous E2 treatment (1, 2) and elevations in serum LH after treatments with aromatase inhibitors (29). The roles of estrogens and ER{alpha} are further supported clinically by the elevated serum FSH levels in an estrogen-resistant patient (30) and in aromatase-deficient humans (31).

The question of AR- vs. ER-mediated negative feedback on serum gonadotropins is further complicated by recent characterizations of a second nuclear ER, termed ERß (32, 33). Although ERß mRNA levels are very low in adult mouse pituitaries (34), there are reports of ERß protein and mRNA in hypothalamic nuclei of both rats and mice (34, 35, 36). Thus, androgens could regulate neuronal activity directly through AR signaling pathways or indirectly through aromatization and activation of either ER{alpha} or ERß signaling pathways.

The recent development of an ER{alpha} knockout (ERKO) mouse line (37) provides a unique opportunity to explore the roles of AR, ER{alpha}, and ERß in regulating hypothalamic and pituitary function. To better define the roles of these steroid receptors in mediating the negative feedback effects of T on hypothalamic GnRH, pituitary gonadotropin gene expression, and serum levels of gonadotropins, we have conducted castration and steroid replacement experiments using male wild-type (WT) and ERKO mice. The findings from our study indicate that both ER{alpha} and AR signaling pathways effectively regulate GnRH content, but that aromatization and activation of ER{alpha} enhance the negative feedback effects of T on serum gonadotropins. In addition, these findings suggest that hypothalamic ERß does not play an obvious role in regulating GnRH content or gonadotrope physiology in adult male mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male WT and ERKO mice (C57BL/6J strain; 10–12 weeks of age) were anesthetized and castrated according to standard surgical protocols at the NIEHS. Castrated (CAST) males were implanted (sc) 2 weeks after surgery with SILASTIC brand capsules (Dow Corning, Midland, MI) containing crystalline T (10–12 mg), DHT (10–12 mg), or E2 (20 µg/capsule dissolved in sesame oil). Intact and CAST control males were implanted with either empty SILASTIC capsules or capsules filled with sesame oil.

Three weeks after implantation, the males were weighed and killed. Blood samples were collected, and pituitaries and hypothalami were frozen on dry ice for subsequent analysis of steady state levels of gonadotropin mRNA and hypothalamic GnRH content. The preoptic-hypothalamic regions were removed as a tissue block, as previously described (38). Seminal fluid was expressed, and the seminal vesicles were weighed to gauge the efficacy of the exogenous androgen treatments. This study was replicated three times, and the samples were assayed as described below. All studies were conducted according to the principles and procedures outlined by the NIH Guidelines for the Care of Experimental Animals and under an approved animal study protocol from the NIEHS animal care and use committee.

Preparation of SILASTIC capsules
Steroid capsules were prepared by cutting SILASTIC tubing (0.62 id x 0.125 od inches) into 15-mm lengths, sealing one end with SILASTIC adhesive, and filling the capsule with either crystalline DHT or T (Steraloids, Wilton, NH) to a length of 1 cm or with 20 µl E2 (Sigma Chemical Co., St. Louis, MO) suspended in sesame oil (1.0 µg/µl). The tubes were then sealed with SILASTIC adhesive. Immediately before implantation, capsules were rinsed using 70% ethanol and washed with sterile saline.

Gonadotropin, GnRH, and steroid RIAs
Serum LH and FSH were quantified by RIA. The assays were performed using the following materials supplied by the NIDDK: 1) for LH, iodination preparation I-9, reference preparation RP-3, and antiserum S-11; and 2) for FSH, iodination preparation I-8, reference preparation RP-3, and antiserum S-11. These RIAs have been previously described and used in the measuring of mouse gonadotropins (39, 40). Fifty-microliter aliquots of sera were assayed in duplicate for both LH and FSH. Due to limited sample volumes, some samples were assayed for FSH as single tubes. In cases where LH and FSH levels were below the limit of detection, the minimum detectable concentration was assigned to that sample. The lower limits of detection for LH and FSH were 0.12 and 1.6 ng/ml, respectively. The intraassay variations were 5% and 9% for the LH assays and 2% and 4% for the FSH assays. Interassay variations were approximately 10% and 5% for LH and FSH assays, respectively.

In samples in which sufficient serum remained after gonadotropin assays, circulating T, DHT, and E2 levels were determined using direct serum RIA kits (Diagnostic Systems Laboratories, Webster, TX). Limited sample volumes prevented measurement of E2, T, and DHT in all treatment groups. Therefore, serum E2 was measured in E2-treated CAST males, and serum T levels were measured in both intact and T-treated CAST males. Serum DHT was measured only in the DHT-treated CAST males. Due to limited sample volumes, some of the samples were assayed as single tubes for steroid levels. The sensitivities of the T, DHT, and E2 assays were 80, 4, and 5 pg/ml, respectively. All samples were assayed within a single assay, and the intraassay variations were approximately 8%, 4%, and 5% for the T, DHT, and E2 assays, respectively.

Hypothalami were sonicated in 0.1 N acetic acid, boiled for 5 min, and centrifuged at 14,000 rpm in a microfuge at 4 C, and the resulting supernatant was lyophilized overnight. The crude protein pellet was saved for subsequent protein determinations and normalization of GnRH values (41). The lyophilized supernatants were resuspended in assay buffer and subjected to RIA for GnRH as described previously (42). Intra- and interassay variations were approximately 6% and 9%, respectively.

RNA isolation and Northern blot analysis
Total pituitary RNA was isolated from single pituitaries using a modification of the Trizol reagent protocol (Life Technologies, Gaithersburg, MD). Briefly, a single pituitary was homogenized in 200 µl Trizol reagent, incubated at room temperature for 5 min, and extracted with 40 µl chloroform. After thorough mixing, the tubes were centrifuged at 14,000 x g for 15 min at 4 C. Glycogen (10 µg/tube) was added to increase the yield of RNA. The aqueous phase was removed, and RNA was precipitated using 1 vol isopropanol for 10 min at room temperature. RNA was centrifuged at 14,000 x g for 15 min at 4 C, washed with 70% ethanol, and resuspended in 40 µl ribonuclease-free water, and the concentration was determined by UV spectrophotometry.

Aliquots of total RNA (1.0 µg) were suspended in 20 µl RNA loading buffer (Ambion, Austin, TX), fractionated on formaldehyde gels, and transferred overnight to Hybond nylon membranes (Amersham, Arlington Heights, IL) using 10 x SSC (1.5 M sodium chloride-0.15 M sodium citrate). RNA was cross-linked by UV exposure in a Stratalinker (Stratagene, La Jolla, CA). Blots were prehybridized for a minimum of 6 h in 12 ml Northern Max hybridization solution (Ambion) containing salmon sperm DNA. Antisense riboprobes for LHß and PL7 were generated using a Maxiscript kit (Ambion), purified on a Nuc-Trap column (Stratagene), counted, and added to the hybridization buffer at 2 x 106 cpm/ml for both LHß and PL7 probes. After an overnight hybridization at 65 C, blots were washed for 30 min at low stringency (2 x SSC-0.1% SDS, at room temperature), followed by a 30-min wash at high stringency (0.1% SSC-0.1% SDS, at room temperature). Blots were exposed to PhosphorImager screens, and mRNA levels were quantified using a PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA). Blots were then exposed to Hyperfilm (Amersham) for autoradiography and photography.

Ribonuclease protection assays (RPA)
Antisense riboprobes for the mouse FSHß and cyclophilin genes were generated using Ambion Maxiscript kits, and RPAs were conducted using a Hybspeed RPA kit (Ambion). Briefly, total RNA (0.5 µg) was ethanol precipitated with 50,000 cpm each of the FSHß and cyclophilin riboprobes, resuspended in 10 µl Hybspeed hybridization buffer (95 C), and incubated at 65 C for 30 min. After hybridization, samples were treated with a 1:250 dilution of ribonuclease A/T1 mix (Ambion) for 30 min at 37 C. At the end of this period, stop solution was added, and the RNA was precipitated for a minimum of 1 h at -80 C. Precipitates were centrifuged at 14,000 rpm for 15 min at 4 C, the supernatant was carefully removed, and the pellets were resuspended in 8 µl RNA loading buffer (Ambion). The samples were heated at 95–100 C for 3 min, placed on wet ice, and electrophoresed on a denaturing, 6% bis-acrylamide gel (8.3 M urea) at 275 V. Gels were fixed twice for 15 min each time in 10% glacial acetic acid-0.5% glycerol and dried overnight before exposure to PhosphorImager screens and Hyperfilm (Amersham).

Statistical analyses
Data for GnRH content, serum LH and FSH, LHß and FSHß mRNA, and steroid levels were analyzed using Levene’s test for homogeneity of variance. All of the variables, except the serum DHT levels, exhibited statistically significant heteroscedascticity. Therefore, although raw data are graphically presented in the figures, all data (except for serum DHT) were log transformed for the statistical analyses. Data were analyzed using two-way ANOVA, and in most cases, significant interactions between genotype and treatment were noted (see figure legends). Post-hoc analyses were conducted using a Student Newman-Keuls test. In all cases, statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Seminal vesicle weights (SVWs) and circulating levels of steroids
To determine whether physiologically relevant levels of steroids were released from the steroid implants, fluid was expressed from seminal vesicles, and wet weights were determined; serum concentrations of steroids were measured by RIA. Castration significantly reduced SVWs and both T and DHT treatments restored SVW to approximately 80% of the values for intact WT and ERKO mice (Fig. 1Go). No effect was observed with E2 treatment. Based on SVWs, there were no obvious genotypic differences in peripheral responses to the androgens.



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  		Figure 1. SVWs in intact, CAST, and steroid-treated WT and ERKO males. SVWs were normalized to body weights (BW). The data are from one of the replicate experiments and are depicted as the mean and SEs for each group. The CAST groups consisted of mice given a blank SILASTIC capsule (BL) or a capsule containing 17ß-estradiol (E), T, or DHT. Sample size ranged from five to seven mice per group. Two-way ANOVA demonstrated significant treatment effects. By Student-Newman-Keuls test: a, P < 0.05 vs. intact WT; b, P < 0.05 vs. intact ERKO; c, P < 0.05 vs. CAST WT; d, P < 0.05 vs. CAST ERKO.

 
Serum E2 levels in E2-treated CAST WT and ERKO males were 12.7 ± 2.3 pg/ml (n = 7) and 13.0 ± 2.3 pg/ml (n = 11), respectively. These values are comparable to levels previously reported for intact WT and ERKO males (43). Serum T levels in this study were approximately 2-fold higher in intact ERKO males (8.5 ± 2.2 ng/ml; n = 12; P < 0.05) than in intact WT males (3.9 ± 2.4 ng/ml; n = 12). This 2-fold elevation is consistent with a previous report of elevated serum T in ERKO males (44). Although T-treated CAST ERKO males had 2-fold lower serum T levels (3.4 ± 0.5 ng/ml; n = 12; P > 0.05) than T-treated CAST WT males (7.1 ± 2.1 ng/ml; n = 12) or intact ERKO males (see above), these levels were similar to those of intact WT males (see above). Serum levels of DHT were 3.6 ± 0.4 (n = 14) and 3.2 ± 0.3 ng/ml (n = 14) in DHT-treated WT and ERKO males. Coupled with the data on SVW, these data suggest that the levels of E2 and T produced by the SILASTIC capsules were in physiologically relevant ranges.

Hypothalamic GnRH content
As sex steroids are known to exert feedback effects on LHRH neurons (5, 7, 9, 11, 45, 46, 47), hypothalamic GnRH contents were examined. There were no significant differences between mean GnRH contents of intact WT and ERKO males, and castration significantly reduced these concentrations by 42% and 33%, respectively (Fig. 2Go). Treatment with E2 restored GnRH contents in CAST WT males to levels comparable to those found in intact WT males, but it failed to have any effect in CAST ERKO males. However, T treatments restored GnRH content to precastration levels in both WT and ERKO castrates. Interestingly, DHT treatment completely reversed the castration effect on GnRH content in ERKO males, but it only produced a partial reversal (51%) of the castration effect in WT males.



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  		Figure 2. Hypothalamic GnRH contents in intact, CAST, and steroid-treated WT and ERKO males. See Fig. 1Go for group details. Depicted are the mean and SEs for each group. Sample size ranged from 12–20 mice/group. Two-way ANOVA demonstrated no significant genotype effect, but there were significant treatment effects and interactive effects. By Student-Newman-Keuls test: a, P < 0.05 vs. intact WT; b, P < 0.05 vs. intact ERKO; c, P < 0.05 vs. CAST WT; d, P < 0.05 vs. CAST ERKO.

 
LHß mRNA expression and serum LH values
Due to evidence that sex steroids exert strong feedback effects on LHß gene expression and serum levels of LH (1, 2), we measured steady state levels of LHß mRNA and circulating levels of LH. Northern blot analyses revealed that pituitaries from intact, adult male ERKOs contain approximately 2-fold higher levels of LHß mRNA (P > 0.05) compared with those from intact WT males (Fig. 3Go, A and B). Long term castration caused 2.0-fold (P > 0.05) and 1.4-fold (P > 0.05) increases in steady state levels of LHß mRNA in WT and ERKO males, respectively. Treatments of CAST WT males with E2 and T suppressed levels of LHß mRNA to 29% (P < 0.05) and 23% (P < 0.05), respectively, of the levels found in CAST WT controls (Fig. 3Go, A and B), whereas DHT treatments resulted in a 47% increase in LHß mRNA (P > 0.05). Unlike steroid treatments of CAST WT males, neither E2, T, nor DHT treatment of CAST ERKOs resulted in significant changes in steady state levels of LHß mRNA (Fig. 3Go, A and B).



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  		Figure 3. Northern blot analysis of LHß and PL7 mRNA levels in the pituitaries of intact, CAST, and steroid-treated WT and ERKO males. See Fig. 1Go for group details. A, Representative blots of LHß and PL7 are displayed. B, Blots were scanned with a PhosphorImager, and levels of LHß mRNA were normalized to PL7 expression. The sample size was seven mice per group. Two-way ANOVA demonstrated significant genotype, treatment, and interactive effects (P < 0.05). By Student-Newman-Keuls test: c, P < 0.05 vs. CAST WT.

 
Although an earlier study did not find significant elevations of serum LH levels in intact ERKO males (44), we sampled a larger number of males and found that serum LH values were 2-fold higher (P < 0.05) in intact ERKO males (Fig. 4Go). This increased level of serum LH agrees roughly with the 2-fold higher levels of LHß mRNA found in intact ERKO pituitaries. Although castration did not significantly alter LHß mRNA levels in either WT or ERKO males, castration resulted in 5-fold (P < 0.05) and 4-fold (P < 0.05) elevations in serum LH in WT and ERKO males, respectively. Treatments of WT castrates with E2 and T suppressed serum LH to 14% and 6% (P < 0.05), respectively, of the values in CAST WT males (Fig. 4Go). However, E2 treatments of CAST ERKO males failed to suppress serum LH. On the other hand, T treatments of CAST ERKO animals suppressed serum LH by 32% (P > 0.05) relative to levels in CAST ERKO controls. Interestingly, DHT treatment resulted in a 52% increase (P > 0.05) in serum LH values in CAST WT while producing a 58% decrease (P < 0.05) in ERKO males.



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  		Figure 4. Serum LH concentrations in intact, CAST, and steroid-treated WT and ERKO males. See Fig. 1Go for group details. Sample size ranged from 12–22 mice/group. Two-way ANOVA demonstrated significant genotype, treatment, and interactive effects (P < 0.05). By Student-Newman-Keuls test: a, P < 0.05 vs. intact WT; b, P < 0.05 vs. intact ERKO; c, P < 0.05 vs. CAST WT; d, P < 0.05 vs. CAST ERKO.

 
Expression of FSHß mRNA and serum FSH
Steroidal and nonsteroidal testicular factors have been demonstrated to exert feedback effects on FSH synthesis and secretion (1, 2). For this reason, we determined the effects of castration and steroid replacement on steady state levels of FSHß mRNA and serum FSH levels in WT and ERKO males. RPAs revealed no significant genotypic differences in steady state levels of FSHß mRNA in pituitaries from intact WT and ERKO male mice (Fig. 5Go). Castration resulted in a 1.7-fold increase (P < 0.05) in FSHß mRNA levels in WT males (Fig. 5Go), but it failed to have any appreciable effect in ERKO males (Fig. 5Go). Compared with CAST WT controls, E2 treatments of CAST WT males decreased steady state levels of FSHß mRNA by approximately 50% (P < 0.05), whereas T and DHT treatments had no effect. In contrast to WT males, neither castration nor steroid replacement therapy significantly altered FSHß mRNA levels in ERKO males.



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  		Figure 5. Steady state levels of expression of FSHß mRNA in pituitaries from intact, CAST, and steroid-treated WT and ERKO males. See Fig. 1Go for group details. Gels from RPAs were dried and quantified using a PhosphorImager. Levels of FSHß mRNA were normalized to cyclophilin expression. Sample size ranged from 6–19 mice/group. Two-way ANOVA demonstrated significant genotype, treatment, and interactive effects (P < 0.05). By Student-Newman-Keuls test: a, P < 0.05 vs. intact WT; c, P < 0.05 vs. CAST WT.

 
Mean serum FSH values were 20% higher (P > 0.05) in intact ERKO males than in intact WT males (Fig. 6Go). Whereas castration elevated FSHß mRNA only in WT males, it resulted in significant increases (~68%) in serum FSH in both WT and ERKO males. E2 and T treatments of CAST WT males suppressed serum FSH by 36% (P < 0.05) and 47% (P < 0.05), respectively, whereas DHT resulted in a small increase in serum FSH (P > 0.05). Despite the fact that castration significantly increased serum FSH in ERKO males, none of the steroid treatments significantly altered serum FSH in ERKO males (Fig. 6Go). Thus, in WT males, the E2-induced suppression of serum FSH is paralleled by a suppression of FSHß mRNA levels, whereas the T-induced suppression of serum FSH is not accompanied by a similar suppression of FSHß mRNA levels. Furthermore, in ERKO males, the postcastration rise in serum FSH levels without accompanying FSHß mRNA increases is probably due to the removal of testicular inhibins.



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  		Figure 6. Serum FSH concentrations in intact, CAST, and steroid-treated WT and ERKO males. See Fig. 1Go for group details. Sample size ranged from 6–19 mice/group. Two-way ANOVA demonstrated significant genotype, treatment, and interactive effects (P < 0.05). By Student-Newman-Keuls test: a, P < 0.05 vs. intact WT; b, P < 0.05 vs. intact ERKO; c, P < 0.05 vs. CAST WT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sex steroids are critical components of the hormonal feedback loops that regulate serum LH and FSH. The sites for these feedback effects include neurons and cells in the hypothalamus that are in close proximity to the GnRH neurons and the gonadotropes in the pituitary. The ability of these target cells to respond directly to androgens is due to the expression of nuclear AR, whereas the presence of aromatase and nuclear ER allows for the conversion of androgens into estrogens and subsequent activation of ER signaling pathways. Indeed, AR, aromatase and ER{alpha} are coexpressed in many hypothalamic nuclei and in gonadotropes (48, 49). Recent data also indicate that the hypothalamus in rats and mice contains both ERß mRNA and protein (34, 35, 36). To explore the roles of AR, ER{alpha}, and ERß in mediating the negative feedback effects of sex steroids on the hypothalamus and gonadotropes, we examined the effects of castration and steroid replacement on GnRH content, gonadotropin gene expression, and serum gonadotropins in WT and ERKO mice.

Hypothalamic GnRH content
Many studies have documented that GnRH plays a major role in determining both the synthesis and release of LH from gonadotropes. However, the exact roles of T and E2 in regulating GnRH synthesis and secretion remain somewhat controversial. Indeed, studies have demonstrated that castration can increase (5, 6, 7), decrease (50), or have no effect (51) on GnRH mRNA levels, whereas exogenous androgens can either decrease (5, 7) or increase (50) levels of GnRH mRNA. Some of these differences may be explained by differences in experimental designs. In several species, castration leads to a rapid increase in the frequency of GnRH pulses (16, 17, 18, 19), which can be suppressed by T treatment (17, 19). Although GnRH pulse frequencies increase rapidly and persist for weeks after castration, a number of studies have also demonstrated that long term castration results in diminished GnRH content and that either E2 or androgen replacement can restore GnRH contents to intact levels (11, 12, 13). Indeed, one study has demonstrated that in long term castrate guinea pigs, hypothalamic GnRH contents are diminished, whereas GnRH pulse frequencies are increased (16). As GnRH content reflects the net effects of changes in transcription, translation, pro-GnRH processing, GnRH secretion, and degradation, we measured GnRH content as an initial step in addressing the roles of AR, ER{alpha}, and ERß in regulating GnRH neuronal function.

In these studies, we found that GnRH contents were similar in intact WT and ERKO males despite the fact that castrated ERKO males were completely insensitive to exogenous E2. As ERß mRNA levels appear unaltered in ERKO mice (34, 35, 36), this suggests that hypothalamic ERß may not play an obvious role in mediating T or E2 regulation of GnRH content in male mice, and that in the absence of functional ER{alpha}, physiological levels of T can effectively regulate GnRH content directly through AR signaling pathways. However, given the in vitro evidence that ERß and ER{alpha} form heterodimers (52), it is possible that disruption of ER{alpha} has ablated any role of ERß in regulating GnRH content. This possibility will need to be addressed by determining the extent to which ER{alpha} and ERß are colocalized in neurons. Future development of ERß-specific agonists and antagonists and ERß knockout models will also allow further delineation of any roles that ERß may play in regulating the GnRH content in male mice.

The fact that both T and DHT treatments of castrated ERKO mice restored the hypothalamic GnRH content to levels observed in intact ERKO animals confirms that AR signaling pathways can effectively regulate the neuronal GnRH content. Although DHT treatments of WT males were less effective than that of CAST ERKO males, both DHT and T treatments of CAST WT males also resulted in significant elevations of GnRH content. The ability of both E2 and DHT to regulate GnRH content in WT males suggests that both AR and ER{alpha} signaling pathways can regulate hypothalamic GnRH contents in male WT mice. However, the increased effectiveness of DHT in ERKO male mice suggests that ER{alpha} disruption has increased the sensitivities of hypothalamic target cells to androgens. This raises the possibility that the ER{alpha} disruption may have resulted in developmental perturbations that lead to increased sensitivity to DHT.

These experiments measured only GnRH content, which is a reflection of the net rates of pro-GnRH synthesis, processing, and secretion. Thus, it is possible that AR and ER{alpha} receptor systems may regulate different steps in the synthesis and secretion of GnRH that yield similar results in terms of GnRH contents. Indeed, it will be interesting to determine the effects of these treatments on GnRH mRNA levels and GnRH secretion patterns in both WT and ERKO males. Nonetheless, these data are consistent with studies suggesting that both AR and ER{alpha} signaling pathways can regulate hypothalamic GnRH content (5, 11, 12, 13).

LHß mRNA expression and serum LH
In addition to regulating GnRH synthesis and secretion, various investigators have shown that E2 and androgens can suppress LHß mRNA and serum LH levels (1, 2, 20, 21, 23). Despite complete E2 insensitivity, we found that both LHß mRNA and serum LH levels in intact male ERKOs were only 2-fold higher than those in intact WT males. As GnRH contents were comparable, these data suggest that the slightly higher levels of LHß mRNA and serum LH in intact ERKO males stem from estrogen insensitivity at the level of the gonadotrope. However, this hypothesis must be examined by testing for genotypic differences in GnRH release patterns and in vitro challenges of primary pituitary cell cultures with steroids and GnRH. Nonetheless, it is apparent that ER{alpha} disruption results in relatively small elevations in LHß mRNA and serum LH levels in male ERKO mice compared with the 8- to 10-fold increase in LHß mRNA and serum LH levels in ERKO females (53, 54). This suggests that ER{alpha} plays a more dominant role in regulating LHß mRNA and serum LH levels in females, whereas AR signaling pathways are relatively effective in regulating LH synthesis and secretion in intact male mice. This hypothesis is supported by the fact that intact Tfm mice have significantly elevated serum LH values that are not further elevated by castration (27).

Long-term castration of both WT and ERKO males induced small (1.5- to 2-fold) elevations in LHß mRNA levels compared with the effects of castration in male rats (1, 2). The discrepancy between the data from this study and those from rat studies may reflect species differences, as an earlier mouse study reported similar elevations of LHß mRNA after castration (27). Alternatively, as reported for male rats, it is possible that LHß mRNA is rapidly induced after castration and subsequently decreases with increasing length of castration. Interestingly, the large increases in serum LH levels (4- to 5-fold) in castrated male WT and ERKO mice were of much greater magnitude than the increases in LHß mRNA levels. This suggests that alterations in pulsatile GnRH secretion and/or gonadotrope sensitivity to GnRH may be primarily responsible for the elevated serum LH observed in long term castrated WT and ERKO males. This interpretation is consistent with experiments demonstrating that castration leads to increased GnRH pulse frequency and elevated serum LH in several species (16, 17, 18, 19).

Both E2 and T significantly suppressed LHß mRNA and serum LH levels in WT castrates while exerting no significant effects in castrated ERKOs. Thus, it appears that aromatization and activation of ER{alpha} enhance the feedback effects of T on LH synthesis and secretion in male mice. It is possible that the lower serum T levels found in T-treated ERKO mice may account for this genotypic difference in the effects of T. However, the range of serum T levels found in T-treated CAST ERKOs overlapped the range of serum T values in intact ERKOs. Furthermore, despite the fact that intact ERKOs have 2-fold higher serum T levels than intact WT males, serum LH values remain 2-fold higher in intact ERKO males. Thus, the genotypic difference in the ability of exogenous T to suppress serum LH may be primarily due to the ER{alpha} disruption. In theory, ER{alpha} disruption could compromise T-induced suppression of GnRH pulse frequency or alter gonadotrope sensitivity to GnRH and rates of transcription of the gonadotropin subunits. We are presently addressing these possibilities with experiments that examine GnRH pulse frequencies and responses of pituitary cell cultures to steroid and GnRH challenges. Studies have suggested that direct effects of E2 and T on gonadotropes may include the regulation of GnRH receptor numbers (55, 56, 57), intracellular responses to GnRH (56, 57), or transcriptional activity of gonadotropin subunit genes (23, 58). It will be interesting to determine the extent to which ER{alpha} vs. AR signaling pathways regulate GnRH receptor levels, basal levels of transcription of the {alpha}GSU or LHß genes, and gonadotrope responsivity to GnRH.

Despite data indicating that aromatization and activation of ER{alpha} greatly enhance the feedback effects of T on LHß mRNA and serum LH levels, it is unclear why T treatments of CAST ERKO males did not suppress serum LH closer to the levels found in intact ERKO males. Some studies have demonstrated that the duration of castration and steroid therapy or steroid dosages may influence the magnitudes of change in LHß mRNA and serum LH responses (1). Thus, it is possible that different lengths of castration and T treatments or different dosages of T might result in a more robust suppression of serum LH in ERKO mice. An intriguing possibility is that AR signaling pathways may suffice to maintain negative feedback control over serum LH levels, but that ER{alpha} signaling pathways are more potent and, therefore, are better able to reinstate negative feedback control over highly elevated serum LH levels found in CAST males. Finally, it is also possible that T was not as effective in male ERKOs because, in the absence of functional ER{alpha}, nonsteroidal testicular factors may play an enhanced role in mediating LH synthesis and secretion.

Based on experiments demonstrating that exogenous DHT suppressed GnRH mRNA levels (7) and serum LH in rats (24), and that serum LH is elevated in Tfm mice (27), we had anticipated that DHT would effectively suppress serum LH in both WT and ERKO males. Surprisingly, we found that DHT treatments consistently resulted in small increases in serum LH and LHß mRNA levels in CAST WT males while significantly suppressing serum LH in CAST ERKO males. Furthermore, DHT was also less effective in raising GnRH content in CAST WT males. It is possible that DHT failed to suppress serum LH levels in CAST WT because of differences in experimental design (chronic castration and DHT treatments) or because of a pharmacological DHT effect. Nonetheless, these possibilities do not address the observation that identical DHT treatments exerted opposite effects on serum LH levels in male WT and ERKO mice. Perhaps the most parsimonious explanation of this phenomenon would be genotypic differences in metabolism and clearance that resulted in different levels of circulating DHT. However, RIA confirmed that serum DHT levels were similar in DHT-treated WT and ERKO males. Thus, it appears that the absence of functional ER{alpha} has altered hypothalamic and/or pituitary sensitivities to DHT. Such altered sensitivities may be due to developmental changes in patterns of AR expression, expression of steroid-metabolizing enzymes, or developmental selection for cells that are more sensitive to androgens. It is also possible that ER{alpha} disruption has simply increased the availability of transcription factors common to both ER{alpha} and AR signaling pathways. Recent studies indicate no obvious genotypic difference in the levels of hypothalamic AR found by immunohistochemistry (59) or in the levels of AR mRNA in the pituitaries of WT and ERKO mice (our unpublished data). We are presently determining whether ER{alpha} disruption has altered patterns of expression of steroid-metabolizing enzymes in androgen target cells or altered availability of transcription cofactors common to both ER{alpha} and AR signaling pathways.

Although the ERKO mouse has provided some interesting insights into the nuclear receptor systems mediating the feedback effects of T on serum LH, it is important to recognize the possibility that ablation of ER{alpha} signaling pathways has led to the development or enhancement of alternative feedback mechanisms. Indeed, the genotypic differences in the effects of DHT suggest that this may have occurred and that, therefore, ER{alpha} signaling pathways may play a more important role in WT mice. However, studies of Tfm mice indicate that serum LH levels are much higher than those in ERKO males and that, unlike ERKO males, castration of Tfm mice does not lead to a further increase in serum LH levels (27). Thus, comparisons of these two models of steroid insensitivity support the general conclusion that AR signaling pathways are more dominant than ER{alpha} signaling pathways in regulating serum LH levels in male mice.

FSHß mRNA expression and serum FSH
The synthesis and secretion of FSH are stimulated by GnRH and activins and suppressed by inhibins and estrogens. However, the feedback effects of T are more complex. For instance, some studies demonstrate that in vivo T treatments can suppress serum FSH (1), whereas other studies illustrate that T can stabilize or stimulate levels of FSHß mRNA, FSH secretion, and serum FSH when endogenous GnRH stimulation is eliminated through the use of GnRH antagonists in vivo (60) or primary pituitary cultures (1). Given the absence of functional ER{alpha} and elevated serum T levels in ERKO males, it was anticipated that FSHß mRNA levels might be elevated in ERKO males. However, FSHß mRNA levels were comparable in intact WT and ERKO mice. This suggests that estrogen insensitivity at the level of the pituitary does not have a significant impact on basal levels of FSHß mRNA in pituitaries of intact ERKO males. In contrast, ER{alpha} disruption in female mice leads to a 7- to 8-fold increase in FSHß mRNA levels (53). This suggests that a sex difference exists in the importance of ER{alpha} in regulating FSHß mRNA synthesis.

Consistent with earlier rodent studies (1, 2), we found that castration increased FSHß mRNA levels in WT males and that E2 treatments suppressed this increase in WT males. The absence of an androgen effect on FSHß mRNA levels agrees with several studies suggesting that androgens may stabilize FSHß mRNA levels (1, 2). In contrast to WT males, FSHß mRNA levels in ERKO males were not significantly altered by any of the treatments, and therefore, it appears that FSHß mRNA levels are relatively static in long term castrated and steroid-replaced ERKO males. However, it is unclear why the large androgen-induced changes in GnRH content did not induce changes in steady state levels of FSHß mRNA. These data suggest that either the changes in hypothalamic GnRH content in ERKO mice are not accompanied by changes in GnRH secretion patterns or, more likely, that ER{alpha}-mediated effects on the gonadotrope are required for GnRH regulation of FSHß mRNA levels.

In agreement with the normal levels of FSHß mRNA in intact ERKO pituitaries, we found that serum FSH levels were only slightly elevated in ERKO males. In addition, castration significantly increased serum FSH levels in both WT and ERKO males, whereas FSHß mRNA levels were increased only in WT males. This indicates that the postcastration rise in serum FSH in ERKO males is independent of changes in steady state levels of FSHß mRNA. Furthermore, as none of the steroid treatments exerted any significant effect on serum FSH levels in ERKO males, we believe that the postcastration rise in serum FSH levels in ERKO males is due to removal of nonsteroidal testicular factors such as inhibin.

Whereas inhibins may play a dominant role in regulating serum FSH levels in ERKO males, it is clear that both E2 and T can effectively suppress serum FSH in WT males. Interestingly, only the E2 treatments of CAST WT males were effective at suppressing postcastration increases in FSHß mRNA, whereas both E2 and T suppressed the postcastration rise in serum FSH. This indicates that mechanisms underlying T suppression of serum FSH in WT males are separate and distinct from the effects of T on steady state levels of FSHß mRNA. It is possible that T treatments stabilized or increased FSHß mRNA levels as reported in rats (1, 60), but suppressed serum FSH in WT males through a reduction in GnRH pulse frequency. However, T treatments elevated GnRH content but failed to suppress serum FSH in male ERKOs. Thus, in WT mice, it is likely that T is aromatized and acts through gonadotrope ER{alpha} to modulate the translation and/or secretion of FSH independently of changes in FSHß mRNA levels. The fact that T treatments failed to suppress serum FSH in ERKO males lends further support to this hypothesis.

In summary, we have employed the ERKO mouse, a genetic model of estrogen insensitivity, to explore the roles of AR, ER{alpha}, and ERß in regulating gonadotropins. The data presented here indicate that ER{alpha} is the predominant signaling pathway involved in mediating E2 suppression of GnRH content, serum gonadotropins, and gonadotropin subunit mRNA expression in male mice. Thus, it does not appear that hypothalamic ERß plays an obvious role in mediating negative feedback regulation of gonadotropins in male mice. Comparisons of serum LH data in this paper with serum LH values in Tfm mice also indicate that AR signaling pathways are the dominant signaling pathways by which T suppresses serum LH in male mice. Indeed, in the absence of functional ER{alpha}, androgens can effectively act through AR signaling pathways to regulate GnRH content, and this may be the predominant pathway by which androgens regulate serum LH in ERKO males. Nonetheless, it does appear that aromatization and activation of gonadotrope ER{alpha} are required to achieve the levels of LHß mRNA and serum LH found in WT males. Interestingly, the ability of both E and T to suppress serum FSH in WT mice is clearly dependent on the presence of ER{alpha}. It also appears that T suppresses serum FSH levels through ER{alpha}-mediated posttranscriptional effects that are independent of steady state levels of FSHß mRNA. Finally, in the absence of functional ER{alpha}, nonsteroidal testicular factors (i.e. inhibins) may effectively regulate serum FSH levels in male mice.


   Acknowledgments
 
We thank Marianna Molina, Todd Washburn, James Clark, Page Myers, and other members of the NIEHS Comparative Medicine Branch staff for their surgical expertise and animal care. Constructs for generating the LHß and FSHß riboprobes were generously provided by Drs. John Nilsson and Malcolm Low, respectively. We also thank Drs. W. Bocchinfuso, E. M. Eddy, and J. Webster for their careful reading and critiques of this manuscript. In addition, we thank Drs. Beth Gladen and Joseph Haseman for statistical advice.

Received March 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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