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Endocrinology, doi:10.1210/en.2001-211342
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Endocrinology Vol. 144, No. 8 3351-3358
Copyright © 2003 by The Endocrine Society

Sex Differences in Estrogen-Dependent Transcription of Gonadotropin-Releasing Hormone (GnRH) Gene Revealed in GnRH Transgenic Mice

Niren R. Thanky, Ruth Slater and Allan E. Herbison

Laboratory of Neuroendocrinology (N.R.T., R.S., A.E.H.), The Babraham Institute, Cambridge CB2 4AT, United Kingdom; and Centre for Neuroendocrinology and Department of Physiology (A.E.H.), University of Otago School of Medical Sciences, Dunedin, New Zealand

Address all correspondence and requests for reprints to: Prof. Allan E. Herbison, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. E-mail: allan.herbison{at}stonebow.otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms through which gonadal steroids exert feedback actions on the activity of the GnRH neurons are not understood. Using a series of GnRH-LacZ transgenic mice we have examined the manner in which gonadal steroids suppress GnRH mRNA expression in male and female mice. The long-term gonadectomy of 5.5-GNZ-3.5 transgenic mice resulted in significant increases in cellular GnRH mRNA expression (P < 0.05) and plasma LH concentrations (P < 0.01) in both sexes. However, cellular levels of LacZ mRNA and ß-galactosidase, which provide an index of GnRH gene transcription, were only elevated in males after gonadectomy. This sexually differentiated response was also observed in mice gonadectomized for 2 wk. Estrogen replacement in gonadectomized males returned transgene expression to intact levels. Experiments in transgenic mice with 3' and 5' deleted GnRH-LacZ constructs revealed that the suppressive influence of estrogen on LacZ transcription in the male required a critical element located between -5.2 and -1.7 kb of the GnRH promoter. These studies show that the suppression of GnRH mRNA expression by estrogen in the male involves a decrease in GnRH gene transcription that is dependent on a distal GnRH promoter element. The same mechanism does not exist in females, indicating that gonadal steroids suppress GnRH mRNA levels in a sexually dimorphic manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GnRH-1 NEURONS of the medial septum and hypothalamus represent the final output neurons of the neuronal network controlling fertility in all mammals. It is well established that gonadal steroid hormones exert critical regulatory feedback actions on the activity of the GnRH neurons (1, 2, 3). Estrogen and progesterone are both believed to be important in generating the fluctuating expression of GnRH that occurs over the estrous cycle in the female rat (3, 4, 5, 6). Although GnRH biosynthesis is thought to be regulated at several levels, including that of gene transcription, mRNA stability and translation (5, 6), the precise mechanisms and pathways used by gonadal steroids to modulate GnRH production, are not fully established (3, 4, 5, 6).

The majority of studies undertaken in the male and female rat indicate that GnRH mRNA expression is suppressed by gonadal steroids at times of LH-negative feedback (3, 5, 6). In vitro experiments (7) have shown that the suppressive influence of estrogen on GnRH mRNA expression can occur in an isolated organotypic slice of the rostral preoptic area (rPOA), suggesting, at least, that distant transsynaptic inputs may not be involved in this process. Whether this negative feedback may involve direct actions of estrogen on the GnRH neuron is not yet clear. However, the recent identification of low levels of estrogen receptor ß (ERß) in rodent GnRH neurons (8, 9, 10) suggests the possibility of direct transcriptional regulation of the GnRH gene by estrogen (11). In vitro studies in GT1 (12, 13) and placental (14) cell lines have shown that estrogen reduces GnRH promoter activity and provided evidence for the direct transcriptional suppression of the GnRH gene by estrogen (13). Together, these in vitro findings represent important evidence supporting the hypothesis that estrogen may bring about the negative feedback decrease in GnRH mRNA levels by suppressing GnRH gene transcription in vivo.

In the present experiments, we have used a series of GnRH-LacZ transgenic mouse lines (15, 16) to determine whether gonadal steroids, acting in a negative feedback manner, do indeed regulate GnRH gene transcription in vivo in the male and female mouse. By evaluating the expression of native GnRH mRNA, as well as LacZ mRNA and its protein product, ß-galactosidase (ß-gal), we have been able to compare GnRH mRNA and GnRH gene transcriptional responses to gonadectomy (GDX) in vivo. We reveal here a robust and unexpected sex difference in the estrogen-dependent regulation of GnRH gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic lines
Five lines of GnRH-LacZ transgenic mice (C57BL6/J x CBA/CA) carrying four different constructs (Fig. 1Go) were used in these studies: 1) homozygous GNZ mice (3252 line) with 5.5 kb of 5', and 3.5 kb of 3', sequence flanking the complete GnRH gene, with a LacZ cassette incorporated between bases encoding amino acids 2 and 3 of the GnRH decapeptide; 2) heterozygous 5.5-GNLZ-3.5 mice (560 line) identical to GNZs, with the exception that they use a nuclear-localized LacZ as reporter; 3) homozygous 5.2-GNLZ-0 mice (481 line), which have no 3' sequence after the transgene insertion site; and 4) two heterozygous 1.7-GNLZ-0 lines (4163 and 4170), which have 1.7 kb of 5' sequence, and no 3', sequence after the LacZ transgene (Fig. 1Go). The production and characterization of the 481 (15), 3252 (16), and 560 (15, 17) lines of mice have been reported previously, whereas the two 1.7-GNLZ-0 lines were produced in a manner identical to our 1.7-GNZ-0 mice (15), with the single exception that they carry a nuclear localized LacZ reporter rather than the standard LacZ.



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  		FIG. 1. Schematic diagrams showing the different GnRH-LacZ constructs carried by the various GnRH transgenic mice employed in the study. nls, Nuclear localizing signal; pA, polyadenylation signal.

 
Experimental groups
Mice were bred and housed at The Babraham Institute, under conditions of 12 h of light (lights on at 0700 h), with constant access to food and water, and were treated in accordance with UK Home Office requirements under projects 80/972 and 80/1005.

Experiment 1.
Adult male and female GNZ mice (Fig. 1Go) were gonadectomized or given sham surgery, which consisted of skin incision and suturing, under Avertin anesthesia (2% tribromoethanol and 2-methylbutan-2-ol in 10% ethanol; 0.1 ml/20 g body weight, ip) at 2–3 months of age (n = 5–6 per group). Mice were then killed 6 wk later by cervical dislocation and decapitation. Brains were rapidly dissected and stored at -80 C until used for LacZ mRNA in situ hybridization experiments and blood collected for LH RIA. The 6-wk period of GDX ensures that circulating gonadal steroids concentrations, and their effects, have had sufficient time to decline (5, 18).

Experiment 2.
Three groups of adult male and female 5.5-GNLZ-3.5 mice (Fig. 1Go; n = 5–6 per group) were used; two were gonadectomized, and one was given sham surgery as detailed above. Four weeks later, one of the gonadectomized groups was administered estradiol benzoate (1 µg in 100 µl ethyl oleate, sc; Intervet, Cambridge, UK), and the other was administered vehicle (100 µl ethyl oleate, sc; Fluka AG, Buchs, Germany), on designated d 0. Injections were then repeated on d 5 and 10, and mice were killed on the morning (1000–1200 h) of d 14, at a time of LH-negative feedback. This regimen ensured that all mice in the study were killed 6 wk after surgery and attempted to recreate the cyclical fluctuations in estrogen concentrations observed over the normal 4- to 5-d estrous cycle in female mice (19). To enable a strict comparison between the sexes, males were subjected to exactly the same estradiol or vehicle regimen as female mice. Mice were killed by Avertin overdose, and a blood sample was collected and then perfused for immunocytochemistry.

Experiment 3.
Adult male and female [5.2-GNLZ-0, 1.7-GNLZ-0 (line 4163) and 1.7-GNLZ-0 (line 4170)] mice (n = 4–6 per group) were gonadectomized or given sham surgery as above. Six weeks later, mice were killed by Avertin overdose, and a blood sample was collected and perfused for immunocytochemistry.

Experiment 4.
Adult male and female 5.2-GNLZ-3.5 mice (n = 5–6 per group) were gonadectomized or given sham surgery, as above, and were killed 2 wk later by Avertin overdose; a blood sample was collected and perfused for immunocytochemistry.

Immunocytochemistry
All mice were killed by an overdose of Avertin (0.3 ml/20 g body weight), followed by perfusion through the heart with 4% paraformaldehyde, as detailed previously (15), between 1000 and 1200 h. Immediately before perfusion, a blood sample was taken from the right atrium for subsequent LH analysis. Dual-labeling immunocytochemistry for ß-gal and GnRH was undertaken as previously described (15, 17). In brief, one of three sets of 30- or 40-µm-thick coronal sections was processed for ß-gal staining using peroxidase-based, free-floating immunocytochemistry with a polyclonal rabbit ß-gal antisera (1:8,000; ICN Biomedicals, Inc. GmbH, Postfach, Germany; 48 h at 4 C), followed by biotinylated goat antirabbit IgGs (1:200; Vector Labs, Peterborough, UK; 90 min) and Vector Elite avidin-peroxidase substrate (1:100; Vector Labs; 90 min); and the peroxidase was revealed with the nickel-diaminobenzidine tetrahydrochloride chromatin using glucose oxidase. Sections were then treated with 40% methanol/Tris-buffered saline/1% H2O2 and incubated in polyclonal LR1 rabbit GnRH antisera (1:40,000; gift of R. Benoit, McGill University, Canada; 40 h at 4 C), followed by peroxidase-conjugated goat antirabbit IgGs (1:400; Vector Labs; 4 h) and immunoreactivity revealed with diaminobenzidine tetrahydrochloride without nickel. The specificity of both antibodies is well established in the mouse brain (15, 16). Controls consisted of the omission of either ß-gal or GnRH antisera from the immunostaining protocol and resulted in an absence of appropriate staining.

Sections were examined under a Leica Corp. DMRB microscope and individual cells examined at x25–40 objective magnification. For each mouse, three to four sections containing the rPOA, where GnRH perikarya are concentrated, were selected; and the brown GnRH-immunostained cells with and without black nuclear-located ß-gal immunoreactivity were counted in each animal. Values from each animal were then used to determine mean ± SEM values for the groups. Statistical analysis of GDX experiments was undertaken using the Mann-Whitney U test, whereas experiments involving three groups in the intact-GDX ± estrogen replacement were analyzed by one-way ANOVA followed by post hoc Student-Neuman-Keuls tests.

GnRH and ß-gal mRNA in situ hybridization
Mice were killed by cervical dislocation and decapitated. Blood was collected for LH assay, and the brains was rapidly removed and frozen on dry ice. Brains were cut in the coronal plane on a cryostat at 15-µm thickness and GnRH, and ß-gal in situ was undertaken as described previously (16, 20). In brief, a 45-mer oligonucleotide complementary to sequences encoding the last 15 amino acids of exon II was used to detect GnRH mRNA (20), whereas 40- and 45-mer oligonucleotides complimentary to nucleotides 1256–1295 and 2822–2866 of LacZ were used together to detect LacZ mRNA (16). Probes were labeled with 35S to a specific activity of approximately 109 cpm/µg and hybridized to a 1:4 set of rPOA sections, coated with emulsion, and left to develop for 5 d (GnRH) or 8 wk (LacZ) before being lightly counterstained and coverslipped. In each experiment, all of the male brains were processed together and then all of the female brains.

The analysis of GnRH and ß-gal mRNA expression was undertaken as detailed before (20), with a Seescan Image Analyzer (Cambridge, UK), by counting the total number of silver grains found within silver grain clusters overlying individual cells located in the rPOA. A minimum of 30 cells obtained from at least two different sections were analyzed in each animal, and values were combined to provide group means. Statistical analysis was undertaken using the nonparametric Mann-Whitney U test.

RIA for LH
Plasma samples were assayed in duplicate using reagents provided by NIDDK. The assay sensitivity was 0.11 ng/ml, and the intra- and interassay coefficients of variation were 8.6% and 18.6%, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1. Effects of 6-wk GDX on LH, GnRH mRNA, and LacZ mRNA in GNZ mice
GDX induced a robust (P < 0.001) increase in LH secretion in both male and female GNZ mice (Fig. 2AGo). In situ hybridization with the GnRH probe revealed the typical GnRH pattern of hybridized cells within the medial septum and preoptic area, identical to that observed previously in the mouse (8). Studies in our laboratory have shown that the GNZ transgene does not itself encode GnRH mRNA (Slater, R., and A. E. Herbison, unpublished). Cellular silver grain analysis demonstrated that the numbers of silver grains overlying GnRH neurons in the rPOA of gonadectomized animals was significantly (P < 0.05) increased, by approximately 20% and 40%, compared with intact male and female GNZ mice, respectively (Fig. 2BGo). In situ hybridization with the cocktail of two LacZ probes revealed a distribution of hybridized cells (Fig. 3Go) identical to that found in the GnRH in situ hybridization, as well as a low level of signal throughout the lateral septum. Transgene expression in the lateral septum of GnRH-LacZ mice (16) represents cells in which the LacZ transgene continues to be expressed after the endogenous GnRH gene is silenced after birth (21). Quantitative analyses of cellular levels of silver grains overlying hybridized cells within the rPOA demonstrated that GDX significantly (P < 0.05) increased the LacZ mRNA signal, by approximately 40%, in males (Figs. 2CGo and 3Go) but had no effect in female GNZ mice (Fig. 2CGo). Differences in LacZ mRNA levels between sham males and females were not examined, because sections from male and female mice were processed separately. These observations suggest that long-term GDX increases LH secretion and GnRH mRNA levels in both sexes but that this is only accompanied by changes in GnRH gene transcription in the male mouse.



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  		FIG. 2. Effect of 6-wk GDX on (A) serum LH concentrations and cellular levels of (B) GnRH mRNA and (C) LacZ mRNA expression in the rPOA of male and female GNZ mice (n = 5–6, each sex). Results in B and C are presented as mean + SEM silver grains per cell. *, P < 0.05; ***, P < 0.001 vs. sham.

 


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  		FIG. 3. Medium-power photomicrographs showing silver grain clusters over cells located in the rPOA after LacZ mRNA in situ hybridization. A, Sham-gonadectomized male; B, gonadectomized male. Scale bars, 15 µm.

 
Experiment 2. Effects of GDX and estrogen replacement on ß-gal transgene in GNLZ mice
Because the product of LacZ is not posttranscriptionally modified in eukaryotic cells, it is also possible to examine changes in LacZ gene transcription by assessing ß-gal protein levels within tissues or individual cells (20, 22, 23, 24, 25). We therefore undertook the 6-wk GDX experiments on 5.5-GNLZ-3.5 mice that carry exactly the same transgene as GNZ mice but have a nuclear-localized LacZ. This facilitates greatly the analysis of ß-gal expression in GnRH neurons as it is directed to the nucleus of the cell and enables dual GnRH (cytoplasmic)-ß-gal (nuclear) immunocytochemistry (15). In addition, we evaluated, in these experiments, whether estrogen might return transgene expression in gonadectomized mice to normal intact values.

As reported previously (15, 17), dual-labeling immunocytochemistry revealed black nuclear-labeled ß-gal immunoreactivity in large numbers of GnRH neurons throughout the GnRH neuronal continuum. GDX resulted in a significant (P < 0.05) (33%) increase in the percentage of GnRH neurons detected to express ß-gal immunoreactivity in male 5.5-GNLZ-3.5 mice but was found to have no significant effect on transgene expression in females (Fig. 4AGo). Treatment with estradiol returned ß-gal expression levels to intact values in male 5.5-GNLZ-3.5 mice (P < 0.05) but had no significant effect on transgene expression in female mice (Fig. 4AGo). GDX had no effect on the total numbers of GnRH-immunoreactive neurons detected in the rPOA (Table 1Go). In both sexes, GDX significantly (P < 0.01) increased circulating LH levels, whereas estradiol administration returned LH levels to intact levels (Fig. 4BGo). These observations demonstrate that changes in ß-gal protein within GnRH neurons, after GDX, parallel that of LacZ mRNA expression and indicate again that only males respond to GDX, with an increase in GnRH gene transcription. For males, the critical gonadal factor restraining gene transcription seems to be estrogen.



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  		FIG. 4. Effect of 6-wk GDX and 2-wk estrogen replacement (EB) on (A) ß-gal protein expression in GnRH neurons and (B) LH concentrations, in male and female 5.5-GNLZ-3.5 mice. Data in A represent mean + SEM percentage of GnRH neurons expressing ß-gal immunoreactivity (n = 6–7 in each group). *, P < 0.05; **, P < 0.01 vs. sham and EB.

 

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  		TABLE 1. Numbers of GnRH-immunoreactive neurons detected per coronal rostral preoptic area brain section in the different transgenic lines and gonadal steroid manipulations

 
Experiment 3. Identification of 3' and 5' regions of the GnRH gene responsible for transcriptional changes after GDX
Dual-labeling immunocytochemistry in 5.2-GNLZ-0 mice revealed that, as in GNZ and 5.5-GNLZ-3.5 animals, GDX increased the percentage of GnRH neurons with detectable ß-gal in males (P < 0.05) but had no effect in female mice (Fig. 5AGo). The numbers of GnRH neurons detected in all four groups of 5.2-GNLZ-0 mice were not different (Table 1Go). Circulating LH levels were elevated by GDX in both sexes (P < 0.01; Fig. 5BGo).



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  		FIG. 5. Effect of 6-wk GDX on (A) ß-gal protein expression in GnRH neurons and (B) LH concentrations, in male and female 5.2-GNLZ-0 mice. Data in A represent mean + SEM percentage of GnRH neurons expressing ß-gal immunoreactivity (n = 5–6 in each group). *, P < 0.05; **, P < 0.01 vs. sham.

 
Dual-labeling immunocytochemistry in two independent lines of sham-treated 1.7-GNLZ-0 mice demonstrated that approximately 20% of all GnRH neurons located throughout the GnRH neuronal continuum expressed detectable ß-gal (Fig. 6Go, A and B). A nonsignificant trend toward higher numbers of detectable GnRH immunoreactive cells was evident in gonadectomized mice (Table 1Go). However, in both lines of 1.7-GNLZ-0 mice, GDX was not found to have any significant effect on transgene expression in either male or female 1.7-GNLZ-0 mice (Fig. 6AGo). Circulating LH levels were elevated significantly (P < 0.01) by GDX in both lines and in both sexes (Fig. 6BGo).



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  		FIG. 6. Effect of 6-wk GDX on ß-gal protein expression in GnRH neurons of two independent lines of 1.7-GNLZ-0 mice (4163 and 4170; n = 4–6, each group) and effect on LH concentrations in both 1.7-GNLZ-0 lines combined. Data in A and B represent mean + SEM percentage of GnRH neurons expressing ß-gal immunoreactivity. **, P < 0.01 vs. sham.

 
Experiment 4. Effects of 2-wk GDX on ß-gal expression in 5.5-GNLZ-3.5 mice
Because previous studies have suggested that the mechanisms regulating GnRH gene expression may alter with time after GDX (5), we undertook a final set of studies in 5.5-GNLZ-3.5 mice examined just 2 wk after GDX. As found in the 6-wk GDX experiments, the percentage of GnRH neurons expressing ß-gal was elevated (P < 0.01) by GDX in male mice (Fig. 7AGo). However, GnRH neurons in female mice now showed a significant (P < 0.05) decline in ß-gal expression 2 wk out from ovariectomy (OVX) (Fig. 7AGo). Plasma levels of LH were elevated (P < 0.01) in both sexes after GDX (Fig. 7BGo). The total numbers of GnRH neurons detected were not significantly different among the four groups (Table 1Go).



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  		FIG. 7. Effect of 2-wk GDX on (A) ß-gal protein expression in GnRH neurons and (B) LH concentrations, in male and female 5.5-GNLZ-3.5 mice. Data in A represent mean + SEM percentage of GnRH neurons expressing ß-gal immunoreactivity (n = 6 in each group). *, P < 0.05; **, P < 0.01 vs. sham.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that GDX results in an increase in GnRH mRNA expression in both sexes but that transgene expression, an index of GnRH gene transcription, is elevated only in the male. This male-specific up-regulation of transcription was observed both 2 and 6 wk after GDX but was lost when transgene 5' flanking sequence was reduced to only the first 1.7 kb of the mouse GnRH (mGnRH) promoter. This suggests a critical role for elements in the distal promoter of the GnRH gene in mediating the gonadal steroid suppression of GnRH gene expression. We further show that the increased levels of GnRH gene transcription after GDX, in the male, can be returned to intact values by the administration of estrogen. The importance of estrogen, as opposed to androgens, in suppressing GnRH gene expression in vivo has also been shown in the long-term gonadectomized male rat (18). Together, these results suggest that the mechanisms through which gonadal steroids suppress GnRH mRNA expression in the mouse are sexually dimorphic.

Several groups (20, 22, 23, 24, 25) have taken advantage of the lack of posttranscriptional processing of the LacZ gene to assess patterns of gene transcription in vivo. The results of the present study continue to support this approach. We have found that the steroid-dependent regulation of ß-gal protein parallels that of LacZ mRNA exactly. The ability to assess GnRH gene transcription in vivo through dual-labeling immunocytochemistry in GNLZ mice is a significant technical advantage, compared with conventional in situ hybridization approaches. It is important to discount the possibility that the steroid regulation observed here is in some way an artifact related to the ß-gal protein itself. The lack of changes in ß-gal expression in 1.7-GNLZ-0 mice, which express the same ß-gal as the other GNLZ lines, demonstrates that the steroid-dependent change in ß-gal expression is critically dependent on 5' GnRH regulatory elements of the transgene. This is further emphasized by the observation that the gonadal steroid regulation of LacZ driven by the tyrosine hydroxylase promoter occurs in a different manner from that reported here in GnRH-LacZ transgenic mice (25).

The presence of transgene expression in approximately 20% of GnRH neurons in the 1.7-GNLZ-0 mice reported here is at odds with our earlier report (15) that the GnRH neurons of 1.7-GNZ-0 mice, without a nuclear localization signal on the LacZ, did not express the transgene. The most likely explanation for this difference is that the targeting of the transgene to the nucleus of the GnRH neurons in the 1.7-GNLZ-0 mice has concentrated the low levels of ß-gal, to make it detectable with immunocytochemistry. The observation that the initial 1.7 kb of GnRH gene sequence is capable of generating transgene expression in GnRH neurons, albeit at low levels, is important for our understanding of GnRH gene function in vivo. The present finding agrees well with a recent study by Kim et al.(26) using similar mGnRH-luciferase transgenic mice.

In keeping with the majority of studies undertaken in the rat (3, 5, 6) and monkey (27), we show here that long-term GDX increases cellular GnRH mRNA levels. It is thought that the typically modest elevations in GnRH mRNA expression observed after GDX are part of the steroid-dependent alterations within GnRH neurons that are required to support the increased GnRH biosynthesis and secretion in gonadectomized animals (5). However, rather few studies have compared the gonadal steroid dependence of GnRH gene expression in both sexes in the same experiment. Toranzo et al. (28) reported that GDX increased GnRH mRNA expression in males and female rats, whereas Wray et al. (7) demonstrated that estrogen suppressed GnRH mRNA levels in organotypic slices of both sexes. We now show a similar situation, in the mouse, where GDX significantly increased cellular GnRH mRNA levels in both the male and female. Though subtle sex differences in the degree of the GnRH mRNA response to gonadal steroid manipulation have been observed in all of these studies, the overall pattern of gonadal steroid suppression was identical between males and females.

Despite the similarity in GnRH mRNA response to GDX in male and female mice, we have unexpectedly found that changes in GnRH gene transcription exhibit clear and robust sex differences. Transgene mRNA and protein levels were elevated by long-term GDX in all three independent lines of GnRH-LacZ male mice, whereas no increase was observed in any of the females. Studies in the female rat (29, 30) have shown that significant alterations in GnRH gene transcription occur only in response to estrogen-positive feedback, and that subtle changes in GnRH mRNA content, at other times of the cycle, arise through posttranscriptional mechanisms. Because we have not examined female GnRH-LacZ mice at different times of the estrous cycle and, to our knowledge, no previous experiments have evaluated the role of transcriptional vs. posttranscriptional mechanisms in estrogen-negative feedback in rats, it is not yet possible to determine the species generality of this phenomenon. However, the marked sex differences reported here in transgene expression are at odds with a similar study by Wolfe et al. (31). Using a transgenic mouse in which the human GnRH promoter drives luciferase expression, these authors reported increased luciferase expression in the hypothalamus of both sexes, 4 wk after GDX. Species differences exist in the organization and functioning of the GnRH promoter (32), and it can be difficult to interpret results obtained from a model in which the human GnRH promoter operates within the mouse transcriptional environment.

The sex difference encountered in 6-wk gonadectomized mice was accentuated even further in the 2-wk GDX studies. Whereas males continued to display an up-regulation of transgene expression, female mice now showed a clear suppression of transgene expression after OVX. Earlier studies have suggested that GnRH biosynthesis may fluctuate in the initial weeks after GDX (see Refs. 5 and 18). Our results in males indicate a persistent elevation of GnRH gene transcription, 2 and 6 wk after GDX, but provide evidence for fluctuations in the female; GnRH gene transcription is initially reduced after OVX but, by 6 wk, returns to normal, intact levels. Although operating on a different time frame, these results are reminiscent of the clear sex differences observed in the timing of the increment in LH secretion after GDX in male and female rats (33, 34).

In the male, estrogen has the capacity to reduce GnRH mRNA levels through the suppression of GnRH gene transcription, involving critical 5' regulatory elements located between -1.7 and -5.2 kb of the GnRH promoter. Recent work defining the neuron-specific expression and regulation of the rat GnRH gene has identified two critical elements comprised of a 300-bp enhancer sequence approximately 1.5 kb upstream of a 173-bp minimal promoter (35, 36). Although the importance of these regions is not yet established in the mouse, it seems likely that complex interactions between enhancer and promoter sequences will exist in the regulation of GnRH gene transcription. As such, our present observation suggests that at least one critical regulatory element required for the estrogen-induced suppression of GnRH gene transcription resides in the distal promoter somewhere between -5.2 and -1.7 in the 5' flank of the GnRH gene. This does not exclude the possibility that elements within the proximal promoter may also be involved in this response.

There are at least two explanations for the sex differences encountered in the present studies. First, it is possible that the suppressive effects of gonadal steroids on GnRH mRNA levels in the female occur entirely through posttranscriptional mechanisms, such as changes in GnRH mRNA stability (6), whereas those in the male involve transcriptional control. The second possibility that cannot be excluded is that gonadal steroids suppress GnRH gene transcription in both sexes but that they use different regions of the GnRH gene to do so. For example, if the steroid-dependent mRNA suppression in female mice were to involve GnRH gene sequence outside that encompassed by our largest transgene (5 kb of 5' through to 3.5 kb of 3' sequence), then we would fail to observe any change in transgene expression in this sex. Even so, this would still indicate the presence of a major sex difference, with males requiring an element between -5.2 and -1.7 of the GnRH promoter to suppress GnRH gene transcription, whereas females would not. It is worth noting, however, that the 2-wk GDX experiments indicate that elements within the first 5 kb of 5' flanking sequence seem sufficient to allow a gonadal steroid-dependent up-regulation of GnRH gene expression in female mice.

It is interesting to speculate on the mechanisms through which gonadal steroids may suppress GnRH mRNA expression in a sexually dimorphic manner in the mouse. Several laboratories have provided in vitro evidence for the direct regulation of GnRH gene transcription by estrogen (13, 14, 37), and the recent identification of ERß protein in rat GnRH neurons provides the appropriate transcription factor through which this response may occur (11). However, no sex differences in ERß expression have been reported in the rat (9); and, unless there are substantial species differences or sex differences in critical ERß coactivators, this would not seem to be a strong candidate for mediating the sex differences observed in the present study.

In contrast, one of the most robust sex differences detected so far in GnRH neurons has been that of increased synaptic input in the female (38, 39). Whether this exists in the mouse is not known, although sex differences have been shown recently in the expression of GABAA receptor subunit mRNAs by mouse GnRH neurons (40). Thus, one plausible explanation for the sex differences in gene regulation observed here would be that males and females use different transsynaptic inputs to suppress GnRH biosynthesis. Although both pathways ultimately reduce GnRH mRNA expression, it may be that the female inputs activate second-messenger cascades that impact exclusively on posttranscriptional processing, whereas the male inputs activate pathways that regulate transcription, with or without effects on the transcript. For example, NMDA receptor activation alters GnRH mRNA levels in an exclusively posttranscriptional manner in vivo, whereas the more general activation of protein kinase A or protein kinase C signaling in GT1 cells results in a decrease in both GnRH gene transcription and mRNA stability (6). The idea that gonadal-steroid-negative feedback, directed at the GnRH neurons, may involve multiple different elements operating in different time frames (3) would be compatible with the changes in GnRH gene transcription that we have observed with time after OVX in the female mouse.

In summary, we used GnRH-LacZ transgenic mouse models to examine mechanisms of gonadal-steroid-negative feedback on GnRH gene expression and uncovered an unexpected sex difference in the transcriptional control of the gene. In the male mouse, estrogen directly or indirectly is able to interact with distal promoter elements to suppress GnRH gene transcription. We have found no evidence that the same mechanism exists in the female mouse, suggesting the possibility that gonadal steroids may suppress GnRH mRNA, in this sex, through posttranscriptional mechanisms.

More generally, these findings highlight that, in addition to the well-established sexually differentiated positive feedback effects (41), sexually dimorphic mechanisms are also involved in the negative feedback effects of gonadal steroids on GnRH neurons.


   Acknowledgments
 
Sandra Dye, Jean-Remi Pape, and Michael Skynner are thanked for assistance with these studies. Dr. R. Benoit is thanked for the generous gift of his LR1 antisera, as is Dr. A. F. Parlow for RIA reagents.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council (UK) and the Wellcome Trust.

Abbreviations: ERß, Estrogen receptor ß; ß-gal, ß-galactosidase; GDX, gonadectomy; mGnRH, mouse GnRH; OVX, ovariectomy; rPOA, rostral preoptic area.

Received November 29, 2001.

Accepted for publication April 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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