Department of Molecular Genetics (A.J.P., R.R.B.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Zoology (A.J.P.), University of Melbourne, Victoria 3010, Australia; Division of Endocrinology, Diabetes, and Hypertension (H.K., U.B.K.), Harvard Medical School, Boston, Massachusetts 02115; Departments of Physiology and Pharmacology (P.M.C.), and Cell and Developmental Biology, Oregon Health & Science University, and Oregon National Primate Research Center (P.M.C., J.A.J., D.L.H.), Divisions of Neuroscience, Reproductive Sciences and Research Services, Beaverton, Oregon 97006; and Department of Molecular and Human Genetics (D.W.S., M.J.J.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Richard R. Behringer, Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: rrb{at}mdanderson.org.
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ABSTRACT |
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INTRODUCTION |
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Recently, we reported an ENU mutagenesis screen in mice using a genetically engineered balancer chromosome to isolate recessive mutations on chromosome 11 within a 24-cM inversion defined by Trp53 and Wnt3 that cause developmental defects (3). In this screen, a total of 735 pedigrees were analyzed, yielding 230 recessive mutations, 88 of which mapped to the 24-cM region of the chromosome 11 balancer. The remaining 142 mutant phenotypes were not linked to the Trp53-Wnt3 region. The phenotypes associated with these genome-wide recessive mutations included alterations in growth, morphology, coat, and behavior. This is perhaps not surprising because visible mutant phenotypes are the most easily scored in mice. One class of mutant phenotypes that are less easily scored are those associated with sexual development.
In the last decade, numerous genes that regulate sex determination and differentiation in mice have been identified (4). Classically, the identification of sex reversal mutants in mice has been facilitated by X chromosome-linked coat color mutations that lead to variegation caused by X inactivation (5, 6). X+X males will have coat color variegation, whereas XY females will be nonvariegated. However, in a large-scale recessive mutagenesis screen, these types of mutations will likely be missed because there will be slight alteration in the sex ratios within individual pedigrees, and the affected animals will almost certainly be sterile; therefore, depending upon the goals of the screen, the pedigree will likely be discarded. Whereas complete sex reversal may be relatively hard to detect, partial sex reversal or abnormalities in the differentiation of secondary sex characteristics may be obvious by simple external examination.
During our ENU mutagenesis screen, we identified one pedigree in which a quarter of the males exhibited abnormalities in their external sexual differentiation. Subsequent analysis also revealed sex differentiation defects in females. Further studies revealed hypogonadotrophic hypogonadism. The recessive mutation proved to be unlinked to the chromosome 11 balancer region. Meiotic mapping and candidate gene sequencing identified a point mutation in the GnRH receptor (Gnrhr) gene that produces a mutant receptor incapable of signal transduction. In addition, we identified an autoregulatory feedback loop for Gnrhr in the pituitary but not the testes.
The Gnrhr gene has been intensively investigated in mammals due its key role in both reproductive function and in numerous medical therapies (7). Furthermore, mutations in human GnRHR are known to be a significant cause of hypogonadotrophic hypogonadism without anosmia or adrenal insufficiency (8, 9). This mouse mutant provides a novel model for studying the precise role of this key signaling pathway in vertebrate and human sexual differentiation and reproduction and for the development of effective treatment regimens for human idiopathic hypogonadotrophic hypogonadism (IHH).
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RESULTS |
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Affected males were readily distinguishable from unaffected males by the small size of their genital papilla and reduced genital-anal distance (Fig. 1). The adult internal reproductive tract was prepubescent in appearance with small testes (approximately one tenth the weight of unaffected littermates) located in the abdominal cavity (Fig. 1
). The testicular cords showed an arrest in spermatogenesis, with germ cells blocked at the early to midpachytene stages of meiosis (Fig. 2
). The sizes of the androgen-dependent seminal vesicle, epididymis, and os penis were dramatically reduced in affected males as compared with unaffected littermates (Fig. 1
).
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Hypogonadotropic Hypogonadism
A significant reduction in circulating FSH levels was observed in the affected individuals of both sexes (Fig. 3). Circulating levels of progesterone, testosterone, androstenedione, and dihydrotestosterone were determined. Although it is natural that the various hormones tested vary between males and females, in each case a significant reduction was observed in circulating levels of the hormones in affected mice when compared with unaffected littermates (Fig. 3
).
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A Point Mutation in Gnrhr
Analysis of pooled and individual DNA samples from affected and unaffected mice showed strong linkage for markers on chromosome 5. The peak LOD score was 12.59 at a recombination fraction of 0.03 between marker D5Mit309 (located at 44 cM on chromosome 5) and the mutation. This locus coincides with the Gnrhr gene, a strong candidate for the mutant phenotype. Sequencing of the three coding exons of Gnrhr revealed a T to C transition in exon 1, 350 bp downstream of the ATG start codon. The point mutation causes a leucine to proline amino acid change at position 117 (Fig. 5A). The nucleotide change eliminates a DdeI restriction enzyme site that facilitates genotyping (Fig. 5B
). ClustalW alignment of Gnrhr nucleotide and amino acid sequence from all GenBank listed species indicated that this amino acid is conserved in all vertebrates from fish to humans. The amino acid change occurs at the N-terminal junction of the third transmembrane domain (Fig. 5C
).
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A dominant-negative effect of L117P on wild-type receptor was demonstrated by cotransfection of both forms into COS7 cells. Cotransfection of wild-type and the L117P mutant form significantly lowered wild-type receptor signal transduction ability in a dose-dependent manner, and this was unaffected by the addition of pharmacoperone, IN3. Cotransfection of the E90K mutant receptor with wild-type receptor also showed a dose-dependent decrease in wild-type signal transduction, but this was completely rescued by the addition of IN3 (Fig. 6E).
Identification of a GnRHR Transcriptional Autoregulatory Feedback Loop
We were able to easily amplify the mutant GnRHR cDNA from the pituitaries of homozygous mutant mice by RT-PCR but had difficulties amplifying the wild-type GnRHR cDNA from wild-type mice. This suggested that mutant GnRHR transcript levels may be elevated in mutant pituitaries relative to wild type. Therefore, GnRHR mRNA levels in the testes and pituitary glands of adult wild-type and homozygous mutant males were determined by real-time RT-PCR. GnRHR mRNA levels were found to be consistently 8-fold higher in homozygous mutant pituitaries in comparison with wild-type littermates (Fig. 5D). However, GnRHR mRNA levels in the testis were not significantly different between wild-type and homozygous mutant littermates (Fig. 5D
). The relative level of GnRH mRNA was also assessed in the hypothamus and ovaries of three adult mutant and three adult wild-type mice by real-time RT-PCR. The levels of GnRH in mutant mice were not significantly different from wild-type levels (data not shown).
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DISCUSSION |
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The ENU-induced point mutation within the Gnrhr gene causes an amino acid change, converting an invariant leucine to a proline. Our in vitro analysis demonstrates that conservation of this amino acid is essential for murine GnRHR function, as cells transduced with GnRHR L117P are unable to bind ligand or transduce signals. This is the first description of a Gnrhr mutation in mice, leading to GnRHR deficiency. This mouse mutant serves as a new animal model for human IHH.
Studies of naturally occurring human mutant GnRHR proteins suggests that most mutations (11 of the 14 known) do not affect the ability of the receptor to function, but rather the ability to fold properly, affecting its subsequent routing to the cell membrane. Treatments with various pharmacological chaperones that promote correct receptor folding and routing result in rescue of receptor function in 11 of the 14 known human GnRHR mutants (11). Rescue has also been shown in a murine ortholog of the human E90K mutant. Chaperone treatment of COS7 cells transfected with the L117P mutant receptor was not able to rescue function with any compound. The proline amino acid substitution would likely produce a break in the helix by forcing a right-angle turn in the peptide; this particular mutant may not be able to be correctly folded even in the presence of chaperones. This is supported by a human mutation that results in a loss of a proline residue in the GnRHR, which similarly cannot be corrected by treatment with rescue agents (14).
Furthermore, we demonstrated that the L117P mutant receptor exerts a dominant-negative effect on wild-type receptor function in a cotransfection assay. Oligomerization of GnRHR in the endoplasmic reticulum is essential for its activation and routing to the plasma membrane. Dominant-negative effects of mutant GnRHRs on wild-type function have been previously demonstrated in humans. This is caused by the intracellular retention of the heterooligomer of the mutant and wild-type receptors and targeting the complex (as a misfolded protein) for destruction in the endoplasmic reticulum, rather than delivering it to the plasma membrane (15). The dominant-negative effect of murine L117P on wild-type receptor function is consistent with the mutant receptor not folding appropriately and not routing to the cell surface, thus affecting the normal routing of wild-type receptors within the same cell. This indicates that the absence of receptor function seen in the L117P mutant is a result of intracellular retention of the mutant receptor.
The levels of circulating reproductive hormones were significantly reduced in Gnrhr mutant mice compared with wild-type or heterozygous littermates. Pituitary immunohistochemistry showed a reduction in the number of gonadotrophs producing FSH and LH. The overall size and histology of the mutant pituitaries were normal. Thus, there may be a reduced number of gonadotrophs or, in the absence of GnRH stimulation, only a subset of gonadotrophs express hormone. It has similarly been noted in the hpg mutant mouse that there is a 30% decrease in the number of pituitary gonadotrophs (16). This level of reduction is insufficient to account for the dramatic drop in reproductive hormone levels, suggesting that, as seen in the Gnrhr mutant, in addition to a reduction in numbers, the levels of hormone production and release from each gonadotroph are also likely reduced.
PRL immunohistochemistry showed similar patterns in wild-type and Gnrhr mutant pituitaries, indicating the mammotrophs were unaffected by the Ghrhr mutation. This is in contrast to the hpg mutant mouse in which PRL levels are significantly changed (10, 17), presumably due to the loss of GAP (GnRH-associated peptide), which forms part of the Gnrh primary transcript from which the bioactive decapeptide, GnRH, is spliced. It has been suggested that GAP either acts to inhibit PRL release from mammotrophs or as a chaperone for GnRHR (18, 19). GAP has also been shown to stimulate FSH and LH secretion from rat primary pituitary cell lines in vitro (20). Although the precise function of GAP remains undetermined, its PRL regulating action must be independent of GnRH-GnRHR signaling because PRL levels remained unaffected in the Gnrhr mutant.
Histological comparison of the Gnrhr and hpg mutant ovaries revealed similarities but also some interesting differences. Hpg mutant ovaries are about a tenth the volume of wild-type ovaries (10), whereas Gnrhr mutant ovaries are only half the size of wild type. Follicles that have undergone early antrum formation were almost never observed in hpg mutant ovaries but are quite frequent in Gnrhr mutant ovaries. This is interesting because hpg mutant ovaries develop large follicles, of a similar size to those observed in the Gnrhr mutant ovaries; however, antrum formation is not induced. This difference may be due to variation in circulating FSH levels between the two mutant mice. FSH levels were undetectable in hpg mice (10), but low and readily detectable in Gnrhr mutants. Perhaps the persistent levels of FSH in Gnrhr mutant mice are sufficient to induce early antrum formation. Alternatively, the compound effect of reduced basal FSH and LH with perturbation of PRL levels in hpg mutant mice may increase the severity of the ovarian phenotype, because PRL has been shown to have direct effects on folliculogenesis (21, 22, 23, 24).
The interstitium of hpg mutant ovaries is consistently described as atretic or cystic (10); however, the interstitium appears normal in Gnrhr mutant ovaries. Our own histological analysis of hpg mutant ovaries also revealed large cystic structures within the interstitium. However, these structures appeared reminiscent of the ovarian hilus, rather than atretic tissue. Immunostaining for CD34, a marker of vascular endothelium, showed that they were indeed vascular spaces, likely the lymphatics of the hilus. The hilus is not normally obvious as it occupies only a small portion of wild-type ovaries. However, in the hpg mutant ovary, the hilus size appears unaffected and is therefore a prominent structure in the tiny mutant ovary.
The testicular phenotypes of Gnrhr mutant male mice are similar to those reported for hpg mutant males (10, 17), both having intraabdominal testes of severely reduced size with a meiotic block in spermatogenesis. The male urogenital tract and external genitalia are similarly undervirilized. However, the interstitial tissue of the testes of Gnrhr mutant males appears normal in contrast to hpg mutant testes in which the interstitium is described as atretic.
Taken together, these data suggest the hpg mutant mouse has a more severe phenotype than the Gnrhr mutant mouse. This indicates that GnRH has additional roles in the reproductive axis, other than signaling through GnRHR alone. Alternatively, the more severe phenotype of hpg mutant mice could be due to the compound loss of GAP leading to changes in PRL levels, and/or other effects of GAP, in addition to the reduced gonadotropin levels. This hypothesis is supported by the finding that suppression of gonadotropins and PRL in eugonadal men treated for prostatic carcinoma results in a more severe reduction in testicular weight and spermatogenesis than a loss of gonadotropin production alone (25). Although the precise role of PRL in the testis remains to be determined, it has been shown to promote steroidogenesis from the Leydig cells of the testis (26, 27) and to have weak gonadotropin action in the testes of hypothalamo-pituitary-disconnected rams (28). Furthermore, PRL has active roles in the interstitial tissue of both the testis and ovary (21, 22, 23, 24). However, it is important to note that the Gnrhr and hpg mutations have been studied on different genetic backgrounds. As such, each mutation is subject to different genetic modifiers that may also be partially responsible for some of the observed phenotypic differences.
Removal of the negative feedback pathway of FSH and LH on the pituitary leads to a dramatic up-regulation of both GnRH and GnRHR (27, 28, 29). In addition, GnRH can positively regulate its own receptor in vivo (30, 31, 32, 33, 34, 35, 36, 37, 38). Not surprisingly then, in the absence of GnRH in hpg mice, pituitary GnRHR levels are only 30% of wild type (39). Administration of exogenous GnRH to hpg mutant mice increases receptor protein, but only to 50% of wild-type levels, presumably due to the overall reduction in the number of gonadotrophs within hpg mutant pituitaries (39). Surprisingly, we found that Gnrhr mutant pituitaries had an 8-fold increase in Gnrhr mRNA levels compared with wild type. This result may be due to mutant transcripts being more stable than wild-type Gnrhr in the pituitary. Alternatively, this could be due to an autoregulatory feedback mechanism in which the inactive or misrouted GnRHR is causing up-regulation of Gnrhr transcription. This mechanism could act to increase the responsiveness of the gonadotrophs to GnRH by increasing receptor numbers in a situation in which GnRH binding and signal transduction are limiting. Interestingly, levels of testicular Gnrhr mRNA remained unaffected in the mutants. This suggests that the regulation of GnRHR in the testis is different than that in the pituitary and independent of the autoregulatory or hormonal feedback mechanisms.
The phenotypes of the Gnrhr mutant mice mirror the abnormalities found in human cases of IHH as a result of GnRHR mutation (9). Determining the response of Gnrhr mutant mice to exogenous hormone regimens will be important for understanding the pathophysiology of this disease and identification of effective treatments for IHH. Furthermore, this mouse will be a valuable tool for understanding the precise in vivo role of GnRHR signaling, among the few key molecular mechanisms controlling and modulating the vertebrate reproductive axis.
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MATERIALS AND METHODS |
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Histology
Tissues for immunohistochemistry were cut at 7 µm and incubated with primary antibody for LH (anti-rat ßLH-IC-3), FSH (NIDK-anti-fFSH-IC-1), or PRL (goat-antimouse-PRL), obtained from the National Hormone and Pituitary Program, Harbor-UCLA Medical Center (Los Angeles, CA) or CD34 (rat-antimouse-CD34) from Cedarlane (Hornby, Ontario, Canada). Hematoxylin and eosin staining and immunostaining were according to standard methods.
LH- and FSH-immunopositive pituitary cells were quantified by determining the number of immunopositive cells in a field of view from two randomly chosen cross-sections of the anterior pituitary for each sample (seven wild-type and nine mutant pituitaries each for LH and FSH). The data were subject to a standard t test.
Hormone Assays
Circulating levels of FSH were obtained from individual plasma samples from eight male mice (four mutant and four wild type) and 10 female mice (four mutants and six wild-type). Progesterone, testosterone, androstenedione, and dihydrotestosterone levels were obtained from three pooled plasma samples containing four mutants or five wild-type mice of mixed sex. Levels were determined by RIA according to standard methods (40, 41).
Mutation Mapping
Pooled DNA samples from N1F1 intercross mice (18 affected and 18 unaffected males and females) were each typed for 72 Massachusetts Institute of Technology microsatellite markers distributed across the genome, to determine a chromosomal localization. Individual DNA samples were then analyzed for a subset of markers on the identified chromosome (42). Each sample was run in quadruplicate on an ABI Prism 377 DNA analyzer. Data were analyzed using GeneScan version 3.1 (Applied Biosystems, Foster City, CA) and called using Genotyper version 2.1 (Applied Biosystems).
Genotyping
Gnrhr exon 1 was amplified by PCR using primers in the flanking genomic sequence (forward, 5'-CTCCACTCTTGAAGCCTGTCC-3'; reverse, 5'-TCACCATGTTCACACAAATTC-3'). Genotype was determined by DdeI digestion of amplified products and electrophoresis on a 4% NuSieve gel (Cambrex Bioproducts, Baltimore, MD).
Functional Analyses
IP Assay: Full-length cDNA clones encoding wild-type and Gnrhr L117P were subcloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA) and transiently transfected into COS7 cells. The Gnrhr expression vectors included six copies of a myc epitope tag at the amino terminus to facilitate detection of the receptors in the absence of an effective anti-GnRHR antibody. The response of transformed cell lines to a GnRH agonist (Buserelin) was measured by IP accumulation as previously described (13). All assay points were performed in triplicate, and each experiment was repeated at least three times. Western blot analysis was performed to confirm mutant receptor expression. Cell lysates were electrophoresed, blotted, and incubated with 9E10 (anti-c-myc) monoclonal antibody (CRP, Inc., Berkeley, CA). Membranes were then stripped, washed, and reprobed with horseradish peroxidase-conjugated antiactin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Receptor binding was performed by incubating transfected cells with [125I]Buserelin as previously described (13). Rescue of mutant receptor function by treatment with pharmacological chaperones and assessment of the dominant-negative effect of mutant receptors on wild-type function was performed as previously described methods (11, 15). To determine the rescue effects of pharmacological chaperones, COS7 cells were transfected with 95 ng of expression plasmids containing the wild-type or mutant cDNA or 5 ng of empty vector. IP accumulation in response to Buserelin stimulation was measured after exposure to chemical chaperones. The dominant-negative effect of mutant L117P GnRHR on wild-type receptor function was determined with COS7 cells transiently transfected with mutant and wild-type Gnrhr at an 8:1 ratio (40 ng mutant + 5 ng wild-type + 55 ng of vector) or a 19:1 ratio (95 ng mutant + 5 ng wild-type). Receptor function was assessed by IP accumulation in response to Buserelin stimulation.
PCR Quantification of Gnrhr mRNA
Total RNA was extracted from the pituitaries and testes of four mutant and four unaffected littermates at 3 wk of age, using RNeasy mini kits (QIAGEN, Chatsworth, CA) and reverse transcribed with Superscript II (Invitrogen, San Diego, CA). Gnrhr transcript levels were determined by real time PCR in the pituitary and gonads of male mice. Reactions where carried out using the Roche Light Cycler and cyber green master mix kits (Roche Clinical Laboratories, Indianapolis, IN). Gnrhr levels were determined in fold changes, as compared with ß-actin levels using the Roche Light Cycler 3.0 software.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online December 29, 2004
Abbreviations: ENU, N-Ethyl-N-nitrosourea; GAP, GnRH-associated peptide; GnRHR, GnRH receptor; IHH, idiopathic hypogonadotrophic hypogonadism; IP, inositol phosphate; PRL, prolactin.
Received for publication May 6, 2004. Accepted for publication December 20, 2004.
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REFERENCES |
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