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*TETRACYCLINE
Endocrinology Vol. 142, No. 5 2102-2114
Copyright © 2001 by The Endocrine Society

ARTICLES

A Conditional Tetracycline-Regulated Increase in Gamma Amino Butyric Acid Production near Luteinizing Hormone-Releasing Hormone Nerve Terminals Disrupts Estrous Cyclicity in the Rat1

Marie Bilger2, Sabine Heger3, Darrell W. Brann, Alfonso Paredes4 and Sergio R. Ojeda

Division of Neuroscience, Oregon Regional Primate Research Center/Oregon Health Sciences University (M.B., S.H., A.P., S.R.O.), Beaverton, Oregon 97006; and Department of Physiology, Medical College of Georgia (D.W.B.), Augusta, Georgia 30912

Address all correspondence and requests for reprints to: Sergio R. Ojeda, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: ojedas{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gamma amino butyric acid (GABA) is the main inhibitory neurotransmitter controlling LH-releasing hormone (LHRH) secretion in the mammalian hypothalamus. Whether alterations in GABA homeostasis within discrete regions of the neuroendocrine brain known to be targets of GABA action, such as the median eminence, can disrupt the ability of the LHRH releasing system to maintain reproductive cyclicity is not known but amenable to experimental scrutiny. The present experiments were undertaken to examine this issue. Immortalized BAS-8.1 astroglial cells were genetically modified by infection with a regulatable retroviral vector to express the gene encoding the GABA synthesizing enzyme glutamic acid decarboxylase-67 (GAD-67) under the control of a tetracycline (tet) controlled gene expression system. In this system, expression of the gene of interest is repressed by tet and activated in the absence of the antibiotic. BAS-8.1 cells carrying this regulatory cassette, and cultured in the absence of tet ("GAD on"), expressed abundant levels of GAD-67 messenger RNA and GAD enzymatic activity, and released GABA when challenged with glutamate. All of these responses were inhibited within 24 h of exposure to tet ("GAD off"). Grafting "GAD on" cells into the median eminence of late juvenile female rats, near LHRH nerve terminals, did not affect the age at vaginal opening, but greatly disrupted subsequent estrous cyclicity. These animals exhibiting long periods of persistent estrus, interrupted by occasional days in proestrus and diestrus, suggesting the occurrence of irregular ovulatory episodes. Administration of the tetracycline analog doxycycline (DOXY) in the drinking water inhibited GAD-67synthesis and restored estrous cyclicity to a pattern indistinguishable from that of control rats grafted with native BAS-8.1 cells. Animals carrying "GAD on" cells showed a small increase in serum LH and estradiol levels, and a marked elevation in serum androstenedione, all of which were obliterated by turning GAD-67 synthesis off in the grafted cells. Morphometric analysis of the ovaries revealed that both groups grafted with GABA-producing cells had an increased incidence of large antral follicles (>500 µm) compared with animals grafted with native BAS-8.1 cells, but that within this category the incidence of steroidogenically more active follicles (i.e. larger than 600 µm) was greater in "GAD on" than in "GAD off" rats. These results indicate that a regionally discrete, temporally controlled increase in GABA availability to LHRH nerve terminals in the median eminence of the hypothalamus suffices to disrupt estrous cyclicity in the rat, and raise the possibility that similar local alterations in GABA homeostasis may contribute to the pathology of hypothalamic amenorrhea/oligomenorrhea in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL established that the neuronal input to the LHRH neuronal network is provided by both stimulatory and inhibitory neurotransmitters (1, 2, 3). Among these transsynaptic regulatory systems, amino acid neurotransmitters appear to have a major role in the control of LHRH secretion (4, 5, 6). Although glutamate stimulates LHRH release via ionotropic (6) receptors, {gamma} aminobutyric acid (GABA)— the dominant neurotransmitter in inhibitory synapses of the hypothalamus (7)—affects LHRH secretion via two different classes of membrane anchored recognition molecules: GABAA receptors, which are ligand-gated anion channels (8, 9, 10), and GABAB receptors, which are seven-transmembrane-domain receptors negatively coupled to adenylate cyclase via GTP- binding proteins (11, 12, 13, 14).

Experiments in nonhuman primates have shown that GABA acting via GABAA receptors restrain LHRH release during sexual development (15) and that removal of this restraining influence results in increased LHRH secretion (16) and advances the onset of female puberty (17). Removal of an inhibitory GABA tone also appears to play a role in the activation of the preovulatory surge of gonadotropins in the adult rat. Although GABA release in the preoptic area decreases before the LH discharge (18), intrahypothalamic infusion of the amino acid in this region of the brain during the afternoon of proestrus abolishes the surge (19). Conversely, blockade of GABAA receptors at this time advances the timing of the surge (20), suggesting that—as in the monkey—the inhibitory effect that GABA exerts on LHRH secretion is mediated by GABAA receptors. Studies in female rats have shown that this inhibitory capacity only develops around the time of puberty (21, 22, 23).

It appears that, at least in rodents, GABA also acts via GABAB receptors to reduce LHRH secretory activity, as pharmacological activation of these receptors inhibits both the preovulatory LH surge (24), and neurotransmittermediated activation of LHRH release (25, 26). In addition, both receptor systems are involved in mediating the inhibitory actions of GABA on pulsatile LH release (27, 28, 29, 30). However, not always GABA inhibits LHRH secretion, as stimulatory GABAA receptor-mediated effects of GABA on LHRH release are well-documented (28, 31, 32, 33). Although part of this stimulatory capacity appears to be developmentally regulated (28, 31), it also appears to be related to a GABAA receptor-mediated increase in excitatory amino acid release (34), and—perhaps more importantly—to a direct excitatory GABA action on LHRH neurons, as shown in a LHRH neuronal cell line (35, 36). That such a direct excitatory effect may be of physiological importance is suggested by the recent findings that normal LHRH neurons in situ contain the complement of GABAA receptor subunits (37, 38) required for the formation of a functional chloride channel responsive to GABA binding and, as transformed LHRH neurons, respond to GABAA receptor stimulation with depolarization in the presence of appropriate chloride concentrations (39, 40). Of added significance is the observation that, in female rats, the inhibitory and excitatory effects of GABA on LHRH secretion appear to be anatomically segregated, with the inhibitory effects most prominently demonstrated in the preoptic area (18, 28, 30), and the excitatory ones in the medial basal hypothalamus/median eminence (33, 41, 42, 43).

It is, therefore, clear that substantial information exists concerning the involvement of GABA in both the control of puberty and the acute regulation of the preovulatory surge of gonadotropins. In contrast, little—if anything—is known about the overall contribution of the amino acid to the maintenance of adult reproductive cyclicity, and the potential importance that specific hypothalamic sites of action may have for the manifestation of such GABA regulatory effect(s). Such knowledge is important as it may offer new insights into the cell-cell mechanisms underlying human reproductive diseases of central origin, but of unknown etiology, such as hypothalamic amenorrhea/oligomenorrhea. To address this issue, we have used a gene transfer-cell grafting system and a conditional gene expression system to produce, in a temporally controlled fashion, regionally circumscribed increases in GABA release in response to the availability of endogenous precursors within the microenvironment surrounding LHRH neurons. Because much less is known about the consequences that GABA actions on LHRH nerve terminals may have on reproductive function, as compared with those on the preoptic region, we targeted the median eminence for grafting GABA-producing cells. The results show that these genetically modified cells disrupt estrous cyclicity via a mechanism that, initiated by an alteration in LHRH output, ultimately compromises ovarian secretory activity. A partial report of these findings has appeared (44).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female rats of the Sprague Dawley strain (B & K Universal, Fremont, CA) were used in these studies. They were housed in a room with a controlled photoperiod (14-h light, 10-h dark; lights on from 0500–1900 h) and temperature (23-25 C), and were given free access to tap water and pelleted rat chow. The animals were used in accordance with the NIH guide for the Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Research Animal Committee.

Cloning of a glutamic acid decarboxylase (GAD)-67 complementary DNA (cDNA) into a tet-regulatable retroviral vector
To generate cells able to release GABA in a regulatable fashion, we engineered immortalized astrocytes to express GAD-67, one of the two enzymes involved in GABA synthesis (45), under the control of a tetracycline (tet)-controlled gene expression system (46, 47). We selected the GAD-67 gene for transgenic expression, as opposed to GAD-65, because GAD-67 is the isoenzyme that may preferentially respond to tonic transsynaptic regulation (48) and, importantly, is the most critical enzyme in GABA synthesis (49). The tet regulatable system employed uses microbial proteins and microbial DNA response elements to drive expression of mammalian genes in heterologous cells (46). In Escherichia coli, transcription of genes conferring tet resistance is inhibited by a tet repressor. In the presence of tet, the repressor (tetR) does not bind to its operators contained in the promoter of the tet operon, so that transcription is allowed to proceed. Gossen and Bujard fused the active domain of VP-16, a potent transcriptional activator, to tetR, creating a hybrid (tTA, tet-controlled transactivator) that stimulates, instead of inhibiting, minimal promoters containing tet operator (tetO) sequences (46). The promoter PhCMV1-1, which in the present case drives GAD-67 expression, is almost silent in the presence of tet, because the antibiotic prevents binding of tTA to the tetO sequences. Thus, in the presence of tet GAD-67 synthesis is repressed and GABA is not produced even in the presence of glutamate precursor. Conversely, in the absence of tet, GAD-67 synthesis is activated, but GABA can only be produced if glutamate is made available to the cell.

To obtain tet-regulated GAD-67 expression we used the retroviral vector LINX (kindly provided by F. Gage, The Salk Institute, La Jolla, CA). LINX is a Moloney murine leukemia virus-based vector that contains all of the components required for tet regulation in a "tet-off" manner (50), as well as a neomycin resistance selection marker (Fig. 1Go). A 1,972-bp rat GAD-67 cDNA encoding the entire open reading frame of GAD-67 messenger RNA (mRNA) (45, 51) [nucleotides (nt) -42 to 1930; a generous gift from A. Tobin (Department of Biology, UCLA, Los Angeles, CA)] was excised from pBS-SK II by digestion with SacII and HindIII, blunted and cloned into the unique (blunted) ClaI site of LINX located directly downstream of the heptamerized tet operator (TetO) sequence fused to the human cytomegalovirus (CMV) immediate early minimal promoter (PhCMV1-1, Fig. 1AGo). The same vector was recently used by Behrstock et al. (52) to express GAD-65, the isoform of GAD-67, under tetracycline control in the conditionally immortalized astrocytic cell line BAS-8.1 (see below).



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  		Figure 1. A, Diagram of the LINX tetracycline (tet)-regulatable retroviral vector used to generate cells overexpressing the GAD-67 gene. The viral LTR transcribes a bicistronic mRNA that in the presence of an IRES (internal ribosomal entry site) element is translated into two protein: tTA (tet repressor-VP16 fusion gene) and neomycin phosphotransferase (neo). In the absence of tetracycline (-tet), tTA binds to, and activates an heptamerized tetracycline operator sequence (tetO) fused to the human cytomegalovirus (CMV) early immediate minimal promoter (CMV TATA) termed PhCMV*-1, resulting in expression of GAD-67. In the presence of tet (+tet), tTA cannot bind to the tetO sequences and expression levels of the GAD-67 mRNA decrease to those determined by the activity of the basal promoter. B, Outline of the gene transfer and selection procedure employed to generate GABA-producing BAS-8.1 cells. For details see Materials and Methods.

 
Cell culture
Two cell lines were used: mouse BAS-8.1 immortalized astrocytic cells (kindly provided by A. Campagnoni, Mental Retardation Research Center, UCLA, Los Angeles, CA) for expression of the LINX-GAD-67 retroviral construct, and the ecotropic packaging cell line Phoenix-E (a generous gift from G. Nolan, Department of Molecular Pharmacology Stanford University, Palo Alto, CA) for the production of replication-defective transmissible viruses (53). The BAS-8.1 cell line is derived from mouse cortical astrocytes immortalized by stable incorporation of a temperature-sensitive mutant of the SV40 large T antigen oncogene into their genome (54). To select the cells that incorporated the oncogene, the immortalizing tsA58 vector was engineered to contain a gene encoding for puromycin resistance, instead of the neomycin resistance gene. Because the thermolabile T antigen protein is active at the permissive temperature of 33-34 C, the host cells can proliferate at this temperature. Upon switching the cells to 37 C, the mutant protein is inactivated, resulting in cessation of cell proliferation and establishment of their differentiated phenotype. Both the packaging cell line and the BAS -8.1 cells were grown in DMEM (Sigma, St. Louis, MO) containing 10% FCS (HyClone Laboratories, Inc., Logan, UT), penicillin G (100U/ml; Sigma), and streptomycin sulfate (100 µg/ml; Sigma). The BAS-8.1 cells had, in addition, puromycin (3 µg/ml; CLONTECH Laboratories, Inc., Palo Alto, CA) added to maintain expression of the T antigen oncogene under selective pressure. A recent report demonstrated the ability of these cells to express GAD-65 and release GABA under the control of the tetracycline-regulated gene expression system (52).

Transfections and infections
The Phoenix-E packaging cell line was transiently transfected with the LINX-GAD-67 retroviral vector using Lipofectamine (Life Technologies, Inc., Grand Island, NY). The cells were seeded at 300,000 cells per well in a 6-well plate. After 24 h, they were washed with PBS solution (PBS) and 1 ml of a transfection cocktail containing 2 µg/ml LINX-GAD67 retroviral vector and 5 µl/ml Lipofectamine in Optimem medium (Life Technologies, Inc.) was added to each well. After 5 h, the transfection medium was replaced with 2 ml of DMEM-10% FCS. Forty-eight hours after transfecting the Phoenix-E cells, the host BAS-8.1 cells were seeded at 350,000 cells per well in a six-well plate, and fresh media were added to the Phoenix cells. Virus containing media was collected from the Phoenix cells 48–72 h after transfection and was passed through a 45 µm filter. Hexadimethrine bromide (Polybrene, Sigma) was added to the virus-containing medium to a final concentration of 4 µg/ml and the medium was applied to the BAS-8.1 cells at 1 ml per well. After 24 h, fresh cultured medium was added to the cells. Selection with neomycin (CLONTECH Laboratories, Inc.) at 500 µg/ml was started 48 h after infection (Fig. 1BGo).

Cell colonies growing under neomycin selection were isolated, replated, and tested for GAD activity (Fig. 1BGo, see below). The colony showing the highest activity was expanded and transfected with a plasmid constitutively expressing the ß-galactosidase (ß-gal) gene under the control of the CMV promoter (pCMV-SPORT-ß-gal, Life Technologies, Inc.). For selection of ß-gal expressing colonies, the cells were cotransfected with a plasmid encoding the hygromycin gene under the control of the thymidine kinase promoter (pTK-Hygro, CLONTECH Laboratories, Inc.). The plasmid was cotransfected with pCMV-SPORT-ß-gal at a 1/10th ratio and selection was initiated 48 h later by adding hygromycin B at 100 µg/ml to the cultures. Colonies growing under triple selection (puromycin, neomycin and hygromycin) were seeded in duplicate 24-well plates and one of the duplicate wells was stained for ß-gal using the procedure described by Sanes et al. (55).

After selecting several ß-gal expressing colonies, they were isolated and again assayed for GAD activity. The colonies showing the highest GAD activity were then evaluated for GAD-67 mRNA content by RNase protection assay following a 24 h treatment with two different doses of doxycycline (DOXY, 1 and 10 µg/ml). The colony containing the highest level of GAD activity and GAD-67 mRNA content in response to DOXY withdrawal was selected for assessment of GABA release in response to a glutamate challenge, and then used for grafting (Fig. 1BGo).

GAD assay
Native and GAD-67-producing BAS-8.1 cells were trypsinized and transferred to a 1.6 ml microcentrifuge tube in PBS. Following centrifugation at low speed, the cell pellets were collected and frozen at -85 C until assay for GAD activity (45, 56). For the assay, the cell pellets were resuspended in a homogenization buffer containing 60 mM potassium phosphate, 0.5% Triton X-100, and the protease inhibitors, 2-aminoethyl-isothiouronium bromide and phenylmethylsulfonyl fluoride at 1 mM each. Twenty microliters of the cell lysate mixture were then incubated in airtight tubes with 0.1 µCi of 14C-L-Glutamate (NEN Life Science Products, Boston, MA), pH 7.0, in 0.1 mM EDTA, 0.5% Triton X-100, 0.1 mM dithiothreitol, 9.0 mM L-glutamate, 30 mM potassium phosphate, and 0.05 mM pyridoxal-5'-phosphate for 1 h at 37 C in a total volume of 60 µl. The 14CO2 released by the GAD-mediated decarboxylation of 14C- labeled glutamic acid was trapped on filters that had been saturated with hyamine hydroxide and allowed to dry before being placed in the tubes above the reaction ingredients. The reaction was stopped with 10 ml of 10% trichloroacetic acid, and the mixture was incubated for another hour at 37 C. After the final incubation the filters were removed, placed in scintillation vials with ScintiVerse BD solution (Fisher Scientific, Pittsburgh, PA) and counted on a Packard 1500 Tri-Carb liquid scintillation counter. The lysate mixture was then assayed for protein content (Protein Assay, Bio-Rad Laboratories, Inc., Hercules, CA), and the amounts of 14CO2 released were calculated as pmol/mg protein·h.

GABA assay
Cells were seeded at 200,000 per well in a 6-well plate with and without DOXY at 1 µg/ml. Twenty-four hours later, the medium was changed to serum-free, glutamate-free medium (DMEM supplemented with 5 µg/ml insulin and 100 µM putrescine) with and without DOXY at 1 µg/ml. Three days later, the cells were washed with PBS and equilibrated in PBS for 30 min before 200 µM of glutamate was added (57) still in the presence or absence of DOXY. The medium was collected 60 min later and was frozen at -85 C until processed for GABA detection by HPLC. All samples (50 µl, run in duplicate) were derivatized with 5 µl of fluoraldehyde (Orthophthaldehyde, Pierce Chemical Co., Rockford, IL) for exactly 10 min. Then, 10 µl from each sample were injected onto a reverse-phase column (Econosphere C-18, 5 µM, 250 x 4.6 mM) and separated using gradient elution. Standards of known concentrations were treated in exactly the same way to circumvent the problem of adduction during the elution process.

The HPLC system (Beckman Coulter, Inc., Fullerton, CA) consisted of a Model 421 microprocessor, Model 110A pumps, an injector with a 20 µl sample loop and a fluorescence detector (Gilson Specra, Gilson Medical Electronics, Inc., Middleton, WI), coupled to a recording integrator (Beckman Coulter, Inc., Altex Scientific Operation, Berkeley, CA). The fluorescence detector used a standard flow cell, a 7–51 excitation filter and a 3–72 M emission filter. The mobile phase in pump A consisted of 0.1 M NaH2PO4 buffer, pH 6.0 containing 10% acetonitrile; that in pump B contained only acetonitrile. The mobile phase was begun at a flow-rate of 1.0 ml/min at 10%B and increased to 40%B over 30 min at which time an isocratic hold lasting 4.5 min was instituted. At the end of the isocratic period, B was increased to 100% over 1 min and maintained at 100% for an additional 10 min. The column was the re-equilibrated with A-B (90:10) for 10 min. Quantification of sample peaks was accomplished by comparing peak areas with those of known concentrations of standards. The data were expressed as pmol GABA/µl sample.

RNA extraction
Total RNA was extracted using the acid phenol method (58, 59) for the extraction of total RNA from cultured cells, as reported (60).

RNase protection assay
The changes in GAD-67 mRNA levels resulting from exposing BAS-8.1 cells carrying the GAD-67 gene to doxycycline were analyzed by RNase protection assay, using 5 µg of total RNA and a procedure described in detail elsewhere (61). Each RNA sample was simultaneously hybridized to 500,000 cpm of a gel-purified 32P-labeled GAD-67 complementary RNA (cRNA) probe and 5,000 cpm of a 32P-labeled cyclophilin antisense RNA probe to correct for procedural variabilities (61). The GAD-67 cRNA probe was generated by in vitro transcription of a 220-bp cDNA template complementary to nt 303–523 in the GAD-67 mRNA coding region (51). The cyclophilin probe was transcribed from a 158-bp PCR-generated cDNA fragment corresponding to nt 265 to 422 in rat cyclophilin mRNA (62).

Grafting
Native BAS-8.1 cells or cells carrying the GAD-67 gene under the control of the tetracycline-inducible system were grafted into median eminence-medial basal hypothalamus of 28- to 30-day-old female rats using an stereotaxic approach described in detail elsewhere (63). To maintain GAD-67 production inhibited before implantation, some cells were exposed in vitro to DOXY (1 µg/ml) for 3- 5 days before grafting. Cells intended to synthesize GAD-67 were left untreated. On the day of grafting, the cells were harvested and processed for implantation following a procedure previously described (63). Each animal received two injections, each containing 40,000 cells in 1 µl of complete phosphosaline buffer, on both sides of the median eminence, 0.4 mm from the midline, as reported (63).

Evaluation of estrous cyclicity and reproductive competence
Starting on the day of grafting, and throughout rest of the experiment, the rats were provided with drinking water containing 0.5% sucrose or 0.5% sucrose plus DOXY at concentrations of 0.1, 1, or 2 mg/ml. The animals were inspected every afternoon for vaginal opening, starting 2 days after grafting; once the vagina became patent, vaginal lavages were obtained daily for assessment of estrous cyclicity. Seven to 42 days after grafting, the animals were anesthetized with tribromoethanol (2.5 mg/100 g BW) and their brains were perfusion-fixed for histochemical evaluation of the grafted cells (see below). Before perfusion, the uterus and ovaries were dissected out, cleaned of adipose tissue and weighed, and the ovaries were inspected for the presence of corpora lutea. The ovaries were fixed in Kahle’s solution (64), embedded in paraffin, sectioned at 8 µm, and stained with methylene blue for histological examination.

Because cyclicity was disrupted in animals carrying GAD-67 producing cells, the fertility of some of these rats was evaluated by placing them with a fertile male for 5 days at a time on two separate occasions. Following introduction of the male into the cage containing the experimental animals, several parameters were evaluated, including the ability of the animals to carry a pregnancy to term, the interval between the introduction of the male and delivery of pups, and the number and body weight of the pups at birth.

In situ localization of the grafted cells and assessment of their functional status
The brains were fixed for either immunohistofluorescence or in situ hybridization, using procedures described in detail elsewhere (65, 66).

a) Immunohistofluorescence-confocal microscopy. This procedure was employed to determine the localization of the grafted cells in relation to the LHRH nerve terminals of the median eminence. LHRH nerve terminals were identified with polyclonal antibodies HFU60 (67) diluted 1:1,000 and the reaction was developed with a fluorescein (FITC)-labeled goat antirabbit {gamma} globulin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:200), as outlined (38). The BAS-8.1 cells carrying the GAD-67 gene were identified with a monoclonal antibody to ß-galactosidase (Roche Molecular Biochemicals, Indianapolis, IN; 500 ng/ml). The reaction was developed with a Texas Red-labeled goat antimouse {gamma} globulin (Jackson ImmunoResearch Laboratories, Inc.; 1:200). Cell nuclei were detected by staining the sections with Hoechst 33258 (bis-benzimide) (Molecular Probes, Inc., Eugene, OR) at 0.1 µg/ml potassium PBS for 1 min after completion of the immunohistochemical reactions. The immunofluorescence images were acquired with a Leica Corp. TCS- SP laser scanning confocal system (Heidelberg, Germany) and a Leica Corp. IRBE microscope, as previously described (68, 69).

b) Immunohistochemistry/in situ hybridization. This procedure was employed to determine whether the grafted cells were indeed responding to the DOXY treatment with the expected changes in GAD-67 expression. BAS-8.1-GAD-67 cells were identified by their content of immunoreactive ß-gal (using the same monoclonal antibody described above) and their content of GAD-67 mRNA was determined by hybridization histochemistry using the same GAD-67 cRNA probe used for RNase protection assays, but labeled with 35S-UTP.

Ovarian morphology
Paraffin-embedded 8 µm serial sections stained with methylene blue (64) were used to determine the number and size distribution of antral follicles. Every fifth section was imaged on a Carl Zeiss Axioplan (Carl Zeiss, Jena, Germany), using a CoolSnap camera (Roper Scientific, Stillwater, MN). Follicle size was estimated only in follicles containing a visible oocyte nucleus (70, 71) by averaging the minimum and maximum diameters of the follicle. Both these measurements and counting of the follicles were carried out using MetaMorph 4.0 (Universal Imaging Co., West Chester, PA). The presence of follicular cysts and precystic structures was also determined using criteria previously reported (72).

RIAs
Circulating LH and FSH levels were measured by RIA as reported (73). Serum levels of ovarian steroids were measured as described (74).

Statistics
The differences between groups were analyzed by ANOVA followed by the Student-Newman-Keuls’ multiple comparison test for unequal replications. Frequency distributions were analyzed using the x2 test. Percentages were subjected to arc-sine transformation before statistical analysis to convert them from a binomial to a normal distribution (75).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BAS-8.1 cells carrying a LINX-GAD-67 retroviral construct express GAD enzymatic activity in a tetracycline-dependent fashion
BAS-8.1 cells stably transfected with LINX-GAD-67 expressed very low GAD activity in the presence of DOXY (1 µg/ml). Three days after removing the antibiotic from the culture medium, GAD activity (measured by the release of 14CO2 from 1-14C-labeled glutamate) increased severalfold. Figure 2Go depicts the GAD activity in several colonies of genetically modified BAS-8.1 cells cultured in the presence and absence of DOXY. Basal levels of GAD activity were similar in native and DOXY-treated LINX-GAD-67 cells. Removal of the antibiotic led to a striking increase in GAD activity in several colonies. Colonies 6 and 8 were the most active and thus were selected for tagging with a reported gene. We chose the ß-gal gene because its protein product can be readily identified by immunohistochemistry to localize the grafted cells in brain tissue (54). Stable incorporation of a CMV driven ß-gal transgene into their genome resulted in abundant ß-gal expression, as assessed by histochemical detection of the protein (Fig. 2Go, inset).



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  		Figure 2. Detection of GAD activity in BAS-8.1 cells selected in neomycin-containing medium for stable expression of the GAD-67 gene under tet regulatory control. GAD activity was determined by the ability of cell extracts to induce the release of 14C from 1-14C-labeled glutamic acid. Several individual neomycin-resistant colonies were analyzed. Numbers under each pair of columns identify the colony assayed. Colony 8 (arrow) was selected for transfection with a CMV-ß-gal expression plasmid that, once incorporated into the cell genome, results in constitutive expression of the Escherichia coli ß-galactosidase (lacZ) gene. Inset, Histochemical detection of ß-gal in BAS-8.1 cells stably transfected with CMV-ß-gal.

 
LINX-GAD-67 cells express GAD-67 mRNA and release GABA in a tet-dependent fashion
Native BAS-8.1 cells had undetectable levels of GAD-67 mRNA (Fig. 3Go, upper panel) and did not release GABA (Fig. 3Go, lower panel) in the presence of 200 µM glutamate. In contrast, when the LINX-GAD-67 cells selected above were cultured in the absence of DOXY they showed markedly elevated levels of GAD-67 mRNA (Fig. 3Go, upper panel) and responded to glutamate with a substantial release of GABA into the culture medium (Fig. 3Go, lower panel). Addition of DOXY markedly reduced, but did not abolish, GAD-67 mRNA content and GABA release from the modified cells (Fig. 3Go).



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  		Figure 3. Upper panel, Tetracycline-dependent expression of GAD-67 mRNA in BAS-8.1 cells carrying a LINX retrovirus in which transcription of the GAD-67 gene is under the control of the tetracycline-regulatable gene expression system. The cells were grown in the absence or presence of doxycycline (DOXY, 1 or 10 µg/ml) for 24 h, at the end of which total RNA was extracted and analyzed for GAD-67 mRNA by RNase protection assay. UP, Undigested probes; DP, digested probes; CYCLO, cyclophilin. Lower panel, GABA release from BAS-8.1 cells carrying the GAD-67 gene under the control of the tetracycline regulated gene expression system. The cells were cultured in glutamate-free medium for 24 h before adding L-glutamate; 200 µM) to the culture wells. The media were collected 1 h later and their GABA content was determined by HPLC. Numbers above bars are number of wells per group. Vertical bars are SEM. ND, Not detectable.

 
LINX-GAD-67 cells tagged with the ß-gal gene and grafted into the median eminence can be identified by their immunoreactive ß-gal content
Figure 4Go depicts confocal microscope images of LINX-GAD-67 cells grafted into the median eminence of the hypothalamus and stained with monoclonal antibodies to ß-gal 6 weeks after grafting. Panel A shows the presence of ß-gal-positive cells (red) in the vicinity of LHRH nerve terminals (green) in the mid-to-lateral ventral portion of the median eminence. Panel B demonstrates at a higher magnification, and in a more lateral plane, that some of the ß-gal-positive cells establish direct contact with LHRH nerve terminals (arrows). Panel C shows a similar view of this relationship, but in the lateral median eminence, where the bulk of LHRH nerve terminals converge to release their secretory products into the portal system. Panel D shows the presence of ß-gal-containing cells in the tract of the needle used for grafting and that runs parallel to the wall of the third ventricle.



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  		Figure 4. Detection of BAS-8.1 cells grafted near LHRH nerve terminals in the median eminence of the hypothalamus by double immunofluorescence-confocal microscopy. LHRH nerve terminals (green color) were visualized with a rabbit polyclonal antiserum to the LHRH decapeptide and an FITC-conjugated second antibody. The grafted cells (red color) were identified with a monoclonal antibody to ß-galactosidase and a Texas Red-conjugated second antibody. Cell nuclei (blue color) were stained with Hoerscht dye. A, ß-gal-positive cells in the vicinity of LHRH nerve endings in the medio-lateral aspect of the median eminence. B, Higher magnification view of a similar field from another animal. C, ß-gal-positive cells near LHRH nerve endings in the lateral aspect of the median eminence. Notice in B and C the close apposition of some LHRH nerve terminals to ß-gal-positive cells (arrows). D, ß-gal-positive cells (examples denoted by double arrows) along the tract of the needle used for grafting (denoted by arrowheads). 3rd V, Third ventricle. Bars in A–C, 5 µm; bar in D, 10 µm.

 
In vivo treatment with DOXY turns off GAD-67 mRNA expression in LINX-GAD-67 cells grafted into the median eminence of the hypothalamus
LINX-GAD-67 cells grafted into the median eminence of rats not receiving DOXY treatment expressed high levels of GAD-67 mRNA, which were reduced to undetectable values in animals treated with DOXY. Figure 5AGo depicts a darkfield image of the median eminence of an animal not treated with DOXY demonstrating the presence of a focus of GAD-67 mRNA expression in the latero-ventral aspect of the median eminence (white frame). That this hybridizing hot spot corresponds to GAD-67 mRNA expressed in the grafted cells is shown in Fig. 5CGo which demonstrates, at a higher magnification and in a brightfield image, that the hybridization signal (white grains) is present in ß-gal immunopositive cells (brown staining). Figure 5BGo demonstrates the absence of focal hybridization in the median eminence of a rat grafted with GAD-67-producing cells and treated with DOXY. This absence of focal expression was due to undetectable levels of GAD-67 mRNA (white grains) in the grafted cells, identified by their ß-gal immunoreactivity (brown color; Fig. 5DGo).



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  		Figure 5. In vivo regulation of GAD-67 mRNA expression in cells carrying the GAD-67 gene under the control of the "tet off" tetracycline-regulated gene expression system, and grafted near the median eminence of the hypothalamus. In this system, GAD-67 synthesis and GABA release are activated in the absence of the tetracycline analog DOXY ("GAD on"), and repressed in its presence ("GAD off"). A, Darkfield image showing a hot spot of 35S-UTP-GAD-67 cRNA hybridization in the latero-ventral aspect of the median eminence from a female rat grafted with "GAD on" cells. B, Absence of focal 35S-UTP-GAD-67 cRNA hybridization in the median eminence of a rat grafted with BAS-8.1 GAD-67 cells and treated with DOXY (2 mg/ml) in the drinking water to inhibit GAD-67 mRNA expression. C, Brightfield image showing that the hybridization signal shown in A (seen here as white grains) is on grafted cells identified by their content of immunoreactive ß-gal (brown color). D, Brightfield image showing the lack of detectable GAD-67 cRNA hybridization to the ß-gal positive cells (brown color) grafted in the boxed area depicted in C. Bars for A and B, 200 µm; bars for C and D, 50 µm.

 
GABA-producing cells grafted into the median eminence disrupt estrous cyclicity in a tet-dependent manner
The age at vaginal opening was similar in rats grafted with "GAD on" BAS-8.1 cells compared with animals grafted with native BAS-8.1 cells (37.4 ± 0.5 vs. 37.0 ± 0.7 days, n = 8 for both groups). Surprisingly, rats grafted with "GAD off" cells, i.e. given DOXY in the drinking water showed a significant delay in vaginal opening (41.2 ± 1.2 days, n = 8). This delay, however, did not appear to be caused by leakage of the delivery system, i.e. the release of small amounts of GABA by the grafted cells (see Fig. 3Go), but instead was related to the DOXY treatment. Administration of the antibiotic to rats grafted with native BAS-8.1 cells or intact animals resulted in ages at vaginal opening (40.2 ± 2.3 and 39.7 ± 0.6 days, n = 5 for both groups) that were similar to that of rats grafted with "GAD off" cells.

In contrast to the lack of effect of the grafts on the onset of puberty, estrous cyclicity was markedly disrupted in animals receiving a graft of GABA-producing cells. As illustrated in Fig. 6Go, these animals exhibited estrous cycles characterized by prolonged periods of estrus sporadically interrupted by occasional days in proestrus and diestrus. An initial dose of 100 µg DOXY/ml water was not sufficient to reverse this profile toward normal cyclicity. Increasing the dose to 2,000 µg/ml was, however, effective. Statistical analysis of the estrous cycle of each animal demonstrated that animals carrying "GAD on" cells spent a greater (P < 0.01) percentage of time in estrus, and much less time (P < 0.01) in proestrus and diestrus than controls rats grafted with native cells (Fig. 7Go). Turning off GABA release by administration of DOXY in the drinking water fully restored the incidence of each phase of the estrous cycle to the values seen in control animals grafted with native cells (Fig. 7Go).



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  		Figure 6. Disruption of estrous cyclicity in postpubertal rats by tetracyclinedependent activation of GABA release from BAS-8.1 cells carrying the GAD-67 gene under the control of the "tet off" tetracycline regulated gene expression system and grafted into the median eminence of the hypothalamus. Two animals from each group are represented. Control animals were grafted with native BAS-8.1 cells. Notice that estrous cyclicity is restored by the 1,000 and 2,000 µg/ml doses of DOXY by not by the lower dose of 100 µg/ml. P, Proestrus; E, estrus; D1, diestrous day 1; D2, diestrous day 2.

 


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  		Figure 7. Analysis of the alterations in estrous cyclicity caused by the tetracycline-dependent activation of GABA release from BAS-8.1 cells carrying the GAD-67 gene under the control of the "tet off" tetracycline regulated gene expression system and grafted into the median eminence of the hypothalamus. The tetracycline-controlled transgene is designed to activate GAD-67 synthesis (and, therefore, GABA release) in the absence of tetracycline (i.e. no DOXY in the drinking water, "GAD on") and to repress GAD-67 synthesis in the presence of DOXY (2,000 µg/ml, "GAD off"). Animals carrying "GAD on" cells exhibited almost twice as many days in estrus than control rats grafted with native BAS-8.1 cells and 3 to 4 times fewer days in proestrus and diestrus, respectively. Treatment with DOXY to turn off GAD-67 expression restored the incidence of all three stages of the estrous cycle to control values. *, P < 0.1 vs. control and "GAD off" groups; **, P < 0.1 vs. "GAD on" group.

 
Rats grafted with either native BAS-8.1 cells or "GAD off" cells became pregnant within the first 5 days of exposure to a fertile male (n = 5). In contrast, of three rats receiving "GAD on" cells, two become pregnant only after a second 5-day exposure to a male, and the third one failed to become pregnant even after three consecutive 5-day exposure periods. No significant differences in the number of pups/litter were found among the three groups.

GABA-producing cells grafted into the median eminence of the hypothalamus disrupt pituitary and ovarian hormone output in a tet-dependent manner
Circulating gonadotropin and sex steroid levels were measured in sera collected 4–6 weeks after the intrahypothalamic grafting of GABA-producing cells. Although FSH levels were not affected (Fig. 8Go, middle upper panel), animals grafted with "GAD on" cells showed a 50% increase in LH levels, which was obliterated (P < 0.05) by DOXY treatment (Fig. 8Go, left upper panel). As a consequence of these changes, the LH:FSH ratio tended to increase in animals carrying "GAD on" cells with respect to the ratio in rats grafted with BAS-8.1 or "GAD off" cells (Fig. 8Go, right upper panel). Whereas a similar tendency was observed in serum estradiol levels (Fig. 8Go, left lower panel), serum P levels were similar in all three groups of animals (Fig. 8Go, middle lower panel). In contrast to the subtle alterations in LH and estradiol levels, serum androstenedione values were noticeably elevated in rats carrying "GAD on" cells, an increase that was absent in rats grafted with "GAD off" cells (Fig. 8Go, right lower panel).



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  		Figure 8. Serum hormone levels in postpubertal rats grafted in the median eminence of the hypothalamus with BAS-8.1 cells carrying the GAD-67 gene under the control of the "tet off" tetracycline regulated gene expression system. The cells were grafted at 28–30 days of age and the serum was collected 4–6 weeks later for hormone measurements. Control rats (grafted with native BAS-8.1 cells) and "GAD on" rats were provided with 0.5% glucose in the drinking water. "GAD off" rats received DOXY (2 mg/ml) in 0.5% glucose in their drinking water. *, P < 0.05 vs. control and/or "GAD off" groups.

 
Ovarian follicular dynamics is altered in animals carrying GABA-producing cells into the median eminence of the hypothalamus
The mean size of ovarian follicles in the 300–499 µm and >500 µm range was similar in the three groups studied, i.e. rats grafted with native BAS-8.1 cells and rats grafted with "GAD on" or "GAD off" cells (Table 1Go). However, the relative distribution of these two populations was different as both "GAD on" and "GAD off" groups had a significantly greater incidence of >500 µm follicles than control rats grafted with native BAS-8.1 cells (Table 1Go). A more detailed examination of this follicle population in the two groups grafted with GAD-67 synthesizing cells (Table 2Go) revealed that in "GAD on" rats there were more large (>600 µm) than intermediate (500–599 µm) size follicles (37/26) and that in the "GAD off" rats this proportion was reversed, i.e. there were more intermediate than large-size follicles (28/37, P < 0.025 by x2 test). In all three groups a low incidence of follicular cysts and precystic structures (follicle type III) (72) was detected in the population of follicles larger than 500 µm. This incidence was similar in all three groups (6 of 36 follicles in the group receiving BAS-8.1 cells; 6 of 63 in the "GAD on" group; and 4 of 65 in the "GAD off" group).


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  		Table 1. Incidence of medium and large size antral follicles in rats carrying intrahypothalamic grafts of GABA-producing cells under the control of the tetracycline-regulated gene expression system

 

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  		Table 2. Changes in the size distribution of large antral follicles in the ovaries of rats carrying intrahypothalamic grafts of GABA-producing cells under the control of the tetracycline-regulated gene expression system

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results demonstrate that conditional activation of GABA release near LHRH nerve terminals in the median eminence of the hypothalamus disrupts estrous cyclicity and reduces fertility in female rats. A regionally restricted increase in GABA release was attained by grafting immortalized astrocytes genetically modified to express GAD-67, one of the GABA synthesizing enzymes, under the control of the tetracycline-dependent gene expression system. Although in this system activation of GAD-67 synthesis is regulated by exogenous manipulation of tetracycline levels, the formation of GABA itself can only occur if endogenous glutamate levels are sufficiently elevated within the microenvironment where the cells were grafted. Thus, the brain is provided with cells able to synthesize GABA, but actual production of the neurotransmitter requires the availability of endogenous precursors. Because of these characteristics, GABA-synthesizing immortalized astrocytes are currently being tested as vehicles for gene therapy of the central nervous system (52, 57, 76).

The neurotransmitter GABA is synthesized from glutamate via a reaction catalyzed by the enzyme GAD. GAD exists in two forms, encoded by two separate genes, GAD-65 and GAD-67 (45). Deletion of the GAD-65 and GAD-67 genes has, however, demonstrated that GAD-67 is the key enzyme for GABA synthesis, as loss of GAD-67 results in more than 90% depletion of GABA levels in the brain, as compared with no reduction in mice deficient in GAD-65 (49). Because most of neuronal GAD-67 is present as an active holoenzyme (45), GABA synthesis would be expected to increase rapidly in the presence of adequate levels of glutamate. Such a change does, in fact, occur in the intact brain (77) and, as shown in the present study, is also observed in GAD-67-expressing cells challenged with glutamate. Previous studies showed that GAD-67-expressing astrocytes preferentially convert glutamate into GABA (57), and rapidly release GABA in response to glutamate, presumably via activation of reverse GABA transporters (57).

Our results show that when GABA availability to LHRH nerve terminals is increased at the end of the juvenile period, the onset of female puberty is not delayed. A delay would have been predicted by earlier studies demonstrating that a) GABA acts on the median eminence of prepubertal rhesus monkeys to inhibit LHRH release (15, 16), and b) that the ability of GABA to delay the pubertal process in rats becomes established during the juvenile period of development (21, 22, 23). Although several more mundane explanations come to mind (for instance, the cells were grafted too late in development, and/or the amount of GABA produced may have been insufficient to suppress LHRH release), we believe that a more tenable explanation for our findings is that—within the rodent hypothalamus—the inhibitory effects of GABA on LHRH secretion are preferentially exerted in the preoptic region, where the LHRH cell bodies are located (18, 28, 30), instead of the median eminence, which was the area selected for grafting.

Although specific experiments may have to be designed to resolve this issue, the intention of the present study was not to reexamine the effect of GABA on the onset of puberty, but instead, to determine whether a discrete change in GABA availability to LHRH nerve terminals, endogenously driven by the availability of glutamate, the GABA precursor, would disrupt adult reproductive cyclicity. The tet-controlled system we employed may not be useful to study developmental aspects of reproductive neuroendocrine control because of the unexpected side effects observed in animals given tet in the drinking water. These animals exhibited a significant delay in vaginal opening as compared with rats drinking only glucose-containing water. The delay was independent of both the surgical procedure used to graft the cells and the presence of foreign cells in the median eminence, as it was also observed in intact rats receiving DOXY. The delay is more likely related to the animals drinking less water due to the bitter taste of DOXY, a reaction observed despite the presence of glucose in the water. Future studies using the tet-controlled system in developing animals will have to search for alternative means of providing the antibiotic orally. Incorporating DOXY to the pelleted food seems to represent a viable alternative for drug administration (78). However, adult animals may be much less sensitive to the treatment than developing rats, because DOXY-treated postpubertal rats cycle and become pregnant as normally as untreated controls receiving grafts of native BAS-8.1 cells.

The tet off-dependent activation of GABA release from GAD-67 producing cells grafted near the median eminence was strikingly effective in disrupting reproductive cyclicity. The GABA dependency of this disruption was indicated by the ability of DOXY treatment—which turns off GABA release—to normalize the estrous cycle toward a pattern indistinguishable from that observed in rats grafted with native BAS-8.1 cells (which are incapable of producing GABA). Two alternative explanations can be offered for these findings. The first assumes that the tet-dependent, focal increase in GABA production near LHRH nerve terminals inhibits LHRH release from those terminals near the grafted cells. This mode of action would be consistent with the wellestablished inhibitory effects that GABA exerts on LHRH release from the monkey median eminence (15) (reviewed in Ref. 79). However, because in our experiments GABAproducing cells led to an apparent stimulation of the hypothalamic-pituitary-ovarian axis (see below), one would have to assume that, if GABA inhibited LHRH release, there was also a compensatory increase in LHRH release from terminals not accessed by GABA. The second explanation, which we favor, is that GABA actually stimulates LHRH release by a direct action on LHRH nerve terminals (42, 43). Such a direct stimulatory effect is in keeping with the demonstration that the direct actions of GABA on LHRH neurons are of excitatory nature (35, 36, 40, 80), and that exposure of isolated medial basal hypothalamic fragments (which contain LHRH terminals without their perikarya) or the median eminence itself to GABA receptor agonists increases, instead of inhibits LHRH release (42, 43).

Although we did not determine the in vivo LHRH response to GABA-producing cells, measurement of serum pituitary gonadotropins and sex steroids levels suggested that, indeed, the hypothalamic-pituitary-ovarian system in rats carrying "GAD on" cells was tonically activated, instead of depressed, by the focal increase in GABA availability. This activation was evidenced by several parameters including the mildly elevated serum LH and estradiol levels, the clearly increased serum androstenedione values, and the higher incidence of large, steroidogenically active (81) ovarian follicles detected in these animals. It thus appears that the presence of GABA-producing cells near a subpopulation of LHRH nerve terminals in the median eminence alters the pattern of LHRH secretion sufficiently to disrupt reproductive cyclicity and cause: 1) an LH-dependent increase in ovarian steroidogenic output; 2) a relative inability of these higher circulating steroid levels to suppress gonadotropin (and presumably LHRH) secretion (because LH levels are not reduced by the high androstenedione and mildly elevated estradiol levels; and c) a disruption of the central mechanism underlying the preovulatory surge of gonadotropins (because "GAD on" rats ovulate sporadically despite the presence of ovulatory competent (82) antral follicles in their ovaries).

Taken altogether, the present results are consistent with the concept that chronic disruption of GABA homeostasis within the confines of the median eminence of the hypothalamus leads to reproductive dysfunction because of an inappropriate increase in LHRH secretion. The disruption in estrous cyclicity, without complete loss of ovulatory capacity, observed in these animals raises the possibility that similar alterations in GABA homeostasis in the same or other relevant regions of the human hypothalamus may contribute to the genesis of at least a fraction of hypothalamic oligomenorrhea/amenorrhea syndromes.


   Acknowledgments
 
We are grateful to Soshana Behrstock and Allan Tobin (Department of Biology, UCLA, Los Angeles, CA) for providing us with the rat GAD-67 cDNA and for their generous help and advice in the preparation and functional analysis of the GAD-67 retroviral construct), and to Les Dees (Texas A & M University, College Station, TX) for measuring serum gonadotropins. We also thank Diane Hill, Veronica Tapia, and Maria E. Costa for their expert technical assistance, and Pablo Ojeda for performing the morphometric analysis of the ovaries.


   Footnotes
 
1 This work was supported by a grant from NICHD/NIH through cooperative U54 HD18185–16 as part of the Specialized Cooperative Centers Program in Reproduction Research (to S.R.O.), NIH Grants RR00163 for the operation of the Oregon Regional Primate Center, HD-28964 (to D.W.B.), and Eli Lilly & Co. Pediatric Endocrine Fellowship grant (to M.B.). Back

2 Pediatric Endocrinology Research Fellow supported by a grant from the Department of Pediatric Endocrinology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. Present address: Valley Children’s Clinic, 2401 River Road, Eugene, Oregon 97404. Back

3 Pediatric Endocrinology Research Fellow supported by a grant from the European Society for Pediatric Endocrinology (ESPE). Back

4 Graduate student, Biochemistry Graduate Program, Faculty of Chemistry and Pharmaceutical Sciences, University of Chile. Supported by a fellowship from NICHD TW/HD00668 Fogarty International Training and Research in Population and Health grant. Back

Received November 6, 2000.


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