Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m110535200
ISSN1083-351X
AutoresHelen H. Kim, Andrew Wolfe, Geary R. Smith, Stuart A. Tobet, Sally Radovick,
Tópico(s)Reproductive System and Pregnancy
ResumoMolecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (−3446/+23 bp, −2078/+23 bp, and −1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5′-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both −2078 and −1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissuesin vivo. We demonstrate that DNA sequences contained within the proximal −1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to −2078 bp mediate the repression of ovarian GnRH. Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (−3446/+23 bp, −2078/+23 bp, and −1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5′-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both −2078 and −1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissuesin vivo. We demonstrate that DNA sequences contained within the proximal −1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to −2078 bp mediate the repression of ovarian GnRH. Appropriate tissue-specific expression of gonadotropin-releasing hormone (GnRH) 1The abbreviations used are:GnRHgonadotropin-releasing hormonehGnRHhuman GnRH, mGnRH, mouse GnRHrGnRHrat GnRHRLUrelative light unitsPBSphosphate-buffered salineGFPgreen fluorescent proteinLUCluciferase 1The abbreviations used are:GnRHgonadotropin-releasing hormonehGnRHhuman GnRH, mGnRH, mouse GnRHrGnRHrat GnRHRLUrelative light unitsPBSphosphate-buffered salineGFPgreen fluorescent proteinLUCluciferase is critical for establishing and maintaining reproductive competence. It has long been recognized that hypothalamic GnRH controls gonadal steroidogenesis and ovarian follicular development by stimulating the production of gonadotropins from the pituitary. More recently, the presence of extra-pituitary GnRH has been appreciated. Low levels of GnRH expression have been found in peripheral reproductive tissues, such as placenta (1Radovick S. Wondisford F.E. Nakayama Y. Yamada M. Cutler G.B., Jr. Weintraub B.D. Mol. Endocrinol. 1990; 4: 476-480Crossref PubMed Scopus (107) Google Scholar), breast, ovary, and testes (2Dong K.W., Yu, K.L. Roberts J.L. Mol. Endocrinol. 1993; 7: 1654-1666Crossref PubMed Scopus (98) Google Scholar). gonadotropin-releasing hormone human GnRH, mGnRH, mouse GnRH rat GnRH relative light units phosphate-buffered saline green fluorescent protein luciferase gonadotropin-releasing hormone human GnRH, mGnRH, mouse GnRH rat GnRH relative light units phosphate-buffered saline green fluorescent protein luciferase The molecular mechanisms that direct the appropriate tissue-specific expression of the GnRH gene are only beginning to be elucidated. The extremely low levels of expression in the peripheral tissues along with the paucity and scattered distribution of GnRH neurons have limited thein vivo study of GnRH gene regulation. In the adult mouse brain, it has been estimated that GnRH expression is limited to only 800 neurons (3Wray S. Grant P. Gainer H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8132-8136Crossref PubMed Scopus (615) Google Scholar). In the adult, the vast majority of the GnRH neurons are located in the basal hypothalamus and septum, but GnRH neurons have been described along the migratory pathway from the olfactory bulbs, as well as in the cerebral cortex and limbic system (3Wray S. Grant P. Gainer H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8132-8136Crossref PubMed Scopus (615) Google Scholar, 4Schwanzel-Fukuda M. Pfaff D.W. Nature. 1989; 338: 161-164Crossref PubMed Scopus (928) Google Scholar). A similar anatomic organization is found in all mammals (5King J.C. Rubin B.S. Crowley W.F. Conn P.M. Serono Symposia: Modes Of Action Of GnRH And GnRH Analogs. Springer-Verlag, New York1994Google Scholar). Several in vitro studies have used mouse-derived immortalized GnRH-secreting neuronal cell lines to investigate the neuronal expression of the rat and mouse GnRH gene. Transient transfection studies using the rat GnRH (rGnRH) gene promoter identified a 173-bp proximal promoter region (6Eraly S.A. Mellon P.L. Mol. Endocrinol. 1995; 9: 848-859Crossref PubMed Google Scholar) and a 300-bp region located 1.8 kb upstream from the transcription start site that conferred cell-specific expression (7Whyte D.B. Lawson M.A. Belsham D.D. Eraly S.A. Bond C.T. Adelman J.P. Mellon P.L. Mol. Endocrinol. 1995; 9: 467-477Crossref PubMed Google Scholar). Both these sites were reported to be important for the correct neuronal expression of the rGnRH genein vitro (8Nelson S.B. Lawson M.A. Kelley C.G. Mellon P.L. Mol. Endocrinol. 2000; 14: 1509-1522PubMed Google Scholar). These in vitro studies have identified several transcription factors that interact with these promoter regions to regulate rGnRH expression: C/EBPβ (9Belsham D.D. Mellon P.L. Mol. Endocrinol. 2000; 14: 212-228PubMed Google Scholar), GATA (10Lawson M.A. Whyte D.B. Mellon P.L. Mol. Cell. Biol. 1996; 16: 3596-3605Crossref PubMed Scopus (73) Google Scholar,11Lawson M.A. Buhain A.R. Jovenal J.C. Mellon P.L. Mol. Endocrinol. 1998; 12: 364-377Crossref PubMed Scopus (39) Google Scholar), Oct-1 (12Clark M.E. Mellon P.L. Mol. Cell. Biol. 1995; 15: 6169-6177Crossref PubMed Scopus (119) Google Scholar, 13Eraly S.A. Nelson S.B. Huang K.M. Mellon P.L. Mol. Endocrinol. 1998; 12: 469-481Crossref PubMed Scopus (62) Google Scholar), Otx 2 (14Kelley C.G. Lavorgna G. Clark M.E. Boncinelli E. Mellon P.L. Mol. Endocrinol. 2000; 14: 1246-1256Crossref PubMed Scopus (64) Google Scholar), and SCIP/Oct-6/Tst-1 (15Wierman M.E. Xiong X. Kepa J.K. Spaulding A.J. Jacobsen B.M. Fang Z. Nilaver G. Ojeda S.R. Mol. Cell. Biol. 1997; 17: 1652-1665Crossref PubMed Scopus (45) Google Scholar). These two promoter regions are highly conserved between the rat and mouse, andin vitro studies with the mouse GnRH (mGnRH) gene promoter suggest that Oct-1 may also regulate the neuronal expression of the mGnRH gene (16Chandran U.R. Warren B.S. Baumann C.T. Hager G.L. DeFranco D.B. J. Biol. Chem. 1999; 274: 2372-2378Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Ultimately, these in vitro studies are unlikely to reflect the elaborate intricacy of in vivo GnRH gene regulation. In the brain, GnRH neurons are dispersed and are influenced by the growth factors, steroids, and neurotransmitters secreted by the various adjacent cell types. Our laboratory has developed previously anin vivo model to study the regulation of the human GnRH (hGnRH) gene in transgenic mice and localized a cell-specific element between −1131 bp and −484 bp of the hGnRH gene (17Wolfe A.M. Wray S. Westphal H. Radovick S. J. Biol. Chem. 1996; 271: 20018-20023Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A transgenic mouse study identified the critical elements for expression of the mGnRH gene between −2.1 kb and −1.7 kb of the distal 5′-sequence (18Pape J.R. Skynner M.J. Allen N.D. Herbison A.E. Mol. Endocrinol. 1999; 13: 2203-2211PubMed Google Scholar). Interestingly, the human and mouse cell-specific regions bear little homology to each other, and neither share homology with the critical rat promoter regions identified in the in vitrostudies. To reconcile these discrepant observations, we constructed transgenic mice with fragments of the mGnRH gene promoter fused to the luciferase (LUC) reporter gene (19de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2466) Google Scholar). Here we report that the proximal −1005 bp of the mGnRH promoter contains the critical elements for hypothalamic expression of mGnRH. Furthermore, we demonstrate that the DNA sequences contained within the proximal −1005 bp are also sufficient to direct mGnRH expression to the ovary. Our study in the mGnRH-LUC mice is the first to define a region of the GnRH promoter that directs GnRH expression to both neural and non-neural tissues in vivo. Unless otherwise indicated, all chemicals and reagents were obtained from Sigma. Restriction enzymes were obtained from New England Biolabs (Beverly, MA) unless otherwise specified. A GnRH promoter-luciferase construct containing −3446 to +23 bp of the mouse GnRH promoter fused to pSV0aLΔ5′ luciferase was kindly provided by Dr. Donald B. DeFranco (University of Pittsburgh, Pittsburgh, PA). The −2078/+23-bp construct was produced by restriction enzyme digestion with BamHI andBglII to remove the sequences between −3446 bp and −2078 bp of the mGnRH promoter. The resulting DNA was re-ligated to generate a BamHI/BglII site. Appropriate ligation was verified by DNA sequencing analysis. The −1005/+28-bp mGnRH-luciferase construct was generated as aHindIII fragment using PCR and the −3446/+23 mGnRH-pSV0aLΔ5′ construct as template. PCR was performed in a thermocycler (PerkinElmer Life Sciences, GeneAmp PCR System 9600), and reaction mixtures contained 5 units of Taq polymerase (DisplayTaq, Display Systems Biotech, Vista, CA) and 0.5 mmdNTPs. The PCR product was restriction enzyme-digested withHindIII and ligated into a HindIII-linearized, alkaline phosphatase (Roche Molecular Biochemicals)-dephosphorylated pA3LUC reporter vector (20Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar, 21Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). Orientation was checked by sequencing constructs using a primer annealing to the 5′ end of the luciferase gene. For the −3446mGnRH-LUC and −2078mGnRH-LUC DNA constructs, the surrounding plasmid was removed from the mGnRH-luciferase fragment byNdeI and Xmn1 double digestion. For the −1005 mGnRH-LUC construct, PvuI digestion was used to isolate it from the surrounding plasmid. The resulting DNA was electrophoresed, and the linear mGnRH-luciferase DNA fragment was excised from the gel, isolated by electroelution into a dialysis membrane (Invitrogen), and purified on an Elutip-D column (Schleicher & Schuell). Transgenic animals were constructed by the Beth Israel Transgenic Facility by pronuclear injection. Fertilized mouse oocytes from FVB-N mice were injected with the purified linear mGnRH-LUC DNA fragment. The resulting embryos were transferred into pseudo-pregnant foster mothers. Transgenic animals were identified with Southern blot analysis as described previously (17Wolfe A.M. Wray S. Westphal H. Radovick S. J. Biol. Chem. 1996; 271: 20018-20023Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, DNA was isolated from tail snips, and restriction enzyme was digested with EcoRI and separated with gel electrophoresis. DNA was then transferred to GeneScreen Plus hybridization transfer membrane (PerkinElmer Life Sciences). A 32P-labeled 1.2-kb probe for luciferase was used to detect transgenic animals that incorporated the luciferase transgene. For identification of luciferase-expressing transgenic lines, neonatal brains were removed from pups, homogenized, and assayed for the mGnRH-luciferase transgene as described below. All procedures were carried out in accordance with the Animal Care and Use Committees of Children's Hospital, Boston, and The University of Chicago. From adult mice, the hypothalamus was dissected in a single fragment consisting of tissue from 1 mm caudal to the mammillary bodies to a point just anterior of the optic chiasm, 1 mm laterally beyond the lateral aspect of the median eminence, and 3 mm dorsally. The olfactory tissue was dissected to include both olfactory bulbs and the tissue rostral to the hypothalamic section. Small representative sections were taken from the remaining tissues. In the case of the gonads, entire gonads were used due to their small size. Similarly, for examination of neonatal luciferase expression, the entire brain was removed from 2- to 3-day-old pups bearing the mGnRH-LUC transgene. Tissues were placed in 1 ml of lysis buffer (25 mmglycylglycine, 15 mm MgSO4, 4 mmEGTA, 1% Triton X-100, and 1 mm dithiothreitol) and homogenized with a Polytron tissue homogenizer (Brinkmann Instruments). The homogenate was centrifuged at 15,000 × g, and the supernatant was assayed for luciferase activity. Luciferase assays were done using a Lumat LB9501 luminometer (Berthold Systems Inc., Pittsburgh, PA). Samples were injected with 100 μl of 0.75 mm luciferin (Molecular Probes, Eugene, OR), dissolved in lysis buffer, and 100 μl of assay buffer (25 mmglycylglycine, 15 mm MgSO4, 4 mmEGTA, 15 mm KPO4, 3 mmdithiothreitol, and 3 mm ATP), and luminescence was measured for 20 s as relative light units (RLU). For some experiments, RLU were normalized for tissue size by correcting for protein content. Protein assays were done using Bio-Rad reagent and bovine serum albumin standards. Brains and ovaries were fixed in situ by perfusing adult mice with a 10% buffered formalin phosphate solution (Fisher). After perfusion, the brains or ovaries were removed and stored overnight in the perfusion solution with 10–15% sucrose. Frozen sections were obtained using a Leica SM2000R sliding microtome. Brain (30 μm) and ovarian (20 μm) sections were collected in phosphate-buffered saline (PBS). Immunocytochemistry using antibodies specific for luciferase and GnRH peptides was performed to co-localize these peptides in the hypothalamus. All incubations were done on a shaking platform. Brain sections were incubated overnight at 4 °C with goat anti-luciferase antibody (Cortex Biochemical, San Leandro, CA) at a concentration of 1–3 μg/ml diluted in PBS with 0.3% Triton X and 1% bovine serum albumin. Sections were washed in PBS, and luciferase immunoreactivity was amplified by incubating with biotinylated horse anti-goat IgG (Vector Laboratories; Burlingame, CA) at room temperature for 2 h at a concentration of 6.75 μg/ml PBS with 0.3% Triton-X. Sections were washed in PBS and then incubated with streptavidin-conjugated Alexa Fluor 488 (Molecular Probes, Eugene, OR) for 2 h at a concentration of 5 μg/ml PBS. After washing with PBS, sections were incubated overnight at 4 °C with a 1:5,000 dilution of GnRH antibody (LR5 rabbit anti-GnRH decapeptide antibody kindly provided by Robert Benoit) in blocking solution of PBS with 0.3% Triton X and 1% bovine serum albumin. Sections were washed with PBS at room temperature and then were incubated with 3 μl/ml Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 2 h and followed by a PBS wash. For the detection of luciferase protein in the ovaries, ovarian sections were incubated overnight at 4 °C with rabbit anti-luciferase antibody (Cortex Biochemical, San Leandro, CA) at a concentration of 1 μg/ml diluted in PBS with 0.3% Triton-X and 1% bovine serum albumin. Sections were washed in PBS and then incubated with 3 μl/ml Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) for 2 h and followed by a PBS wash. Tissue sections were mounted on slides and coverslipped with vectashield mounting reagent (Vector Laboratories, Burlingame, CA). The edges of the coverslips were then sealed with standard nail polish. The double fluorescence histology sections were analyzed on a Zeiss Axiovert SS100TV inverted fluorescent microscope, and images were captured with a Zeiss video camera with a CCD chip. The Cy3 and Alexa Fluor 488 fluorescences were visualized using the appropriate filters. A DNA construct containing the region from −3446 bp to +23 bp of the mGnRH promoter fused to the luciferase reporter gene was used to generate transgenic mice (−3446mGnRH-LUC). As shown in Table I, Southern blot analysis identified four separate founder lines that incorporated the luciferase transgene. Because luciferase expression is under the control of the mGnRH promoter, mGnRH promoter activity can be detected easily by measuring the luciferase activity in tissue homogenates. To identify founder lines expressing the mGnRH-LUC transgene, whole brain homogenates were obtained from neonatal mice and assayed for luciferase activity. Luciferase activity was measured as RLU. Two −3446mGnRH-LUC founder lines were found to express luciferase. In brain homogenates from 2- to 3-day-old pups, luciferase activity was 22,309 ± 1042 RLU in the lower expressing line and 43,488 ± 4762 in the higher expressing line. Luciferase activity in the founder lines that did not express luciferase (19.3 ± 7.4 RLU) did not differ from background levels.Table IEffect of mGnRH promoter fragment on expression of luciferase transgene in the neonatal mouse brainMouse GnRH-LUC constructNumber transgenic lines generatedNumber luciferase-expressing lines generatedLine with lowest neonatal luciferase activityLine with highest neonatal luciferase activityLuciferase activity in non-luciferase-expressing miceRLURLURLU−3446 mGnRH-LUC4222,309 ± 1,04243,448 ± 4,76219.3 ± 7.4(n = 48)(n = 55)(n = 20)−2078 mGnRH-LUC1781,500 ± 10412,983 ± 59925.2 ± 8.7(n = 2)(n = 4)(n = 77)−1005 mGnRH-LUC637,044 ± 47222,259 ± 5,87229.3 ± 9.1(n = 3)(n = 3)(n = 24)Three different mouse GnRH promoter fragments were fused to the luciferase reporter gene and used to generate transgenic mouse lines. Transgenic lines were identified by Southern blot analysis. Luciferase expression was determined by performing luciferase assays in whole brain homogenates from neonatal mice. Luciferase activity was measured as RLU. Luciferase assays were performed in a total of 506 neonatal mice from the 27 different founder lines. Overall, luciferase activity was detected in 48% (13/27) of founder lines containing the luciferase transgene. The table shows the range of luciferase activity seen in different founder lines generated with the same mGnRH-LUC DNA construct. The mean ± S.E. of luciferase activity found in whole brain homogenates from 2–3-day-old pups is shown for the lowest and highest luciferase-expressing founder lines. Luciferase activity, in brain homogenates from non-luciferase expressing lines, was similar to background levels. Open table in a new tab Three different mouse GnRH promoter fragments were fused to the luciferase reporter gene and used to generate transgenic mouse lines. Transgenic lines were identified by Southern blot analysis. Luciferase expression was determined by performing luciferase assays in whole brain homogenates from neonatal mice. Luciferase activity was measured as RLU. Luciferase assays were performed in a total of 506 neonatal mice from the 27 different founder lines. Overall, luciferase activity was detected in 48% (13/27) of founder lines containing the luciferase transgene. The table shows the range of luciferase activity seen in different founder lines generated with the same mGnRH-LUC DNA construct. The mean ± S.E. of luciferase activity found in whole brain homogenates from 2–3-day-old pups is shown for the lowest and highest luciferase-expressing founder lines. Luciferase activity, in brain homogenates from non-luciferase expressing lines, was similar to background levels. In the mGnRH-LUC mice, the anatomic pattern of luciferase expression is an assay of mGnRH promoter activity. As illustrated in Fig.1 A, a similar anatomic pattern of luciferase expression was found in offspring from both founders that expressed the −3446mGnRH-LUC transgene. Data from three female offspring of founder 1 and four female offspring of founder 2 are shown. As expected, high levels of luciferase activity were seen in the hypothalamus (20,148 ± 6093 and 21,263 ± 8214) and olfactory lobes (10,255 ± 1117 and 15,957 ± 3782) when luciferase expression is under the control of the mGnRH promoter. Luciferase activity was detected in the cortex (2771 ± 1505 and 12,572 ± 4027), cerebellum (893 ± 214 and 3807 ± 1255), and midbrain (668 ± 184 and 1427 ± 773). With the exception of the ovary, which had low levels of luciferase activity (154 ± 24 and 41 ± 13), luciferase levels in the other tissues, including the testes, as shown in Fig.2 A, did not differ from background levels. These two lines of −3446mGnRH-LUC mice demonstrate that −3446 bp of the mGnRH promoter targets luciferase expression appropriately to GnRH-containing regions of the mouse brain.Figure 2Ovarian luciferase expression in −1005mGnRH-LUC mice. Ovarian sections were incubated with rabbit anti-luciferase antibody or nonspecific rabbit IgG. Immunoreactivity was detected by anti-rabbit Cy3, which is seen as redfluorescence. A shows a low power (×100) magnification of a representative ovarian section. Luciferase immunoreactivity is seen specifically within two follicles. B and C show the two luciferase-containing follicles at higher magnification (×200). At this higher magnification, it is clear that Cy3 fluorescence is contained within the ovarian granulosa cells.D and E show similar follicles from an ovarian section incubated with nonspecific rabbit IgG. No specific staining of the granulosa cells is seen. There does, however, appear to be some nonspecific staining of the oocyte.View Large Image Figure ViewerDownload (PPT) To confirm that luciferase expression was confined to GnRH neurons in the hypothalamus, double-labeling immunocytochemistry experiments were performed in the −3446mGnRH-LUC mice. Brain sections from the −3446mGnRH-LUC mice were labeled with antibodies specific for luciferase and GnRH peptides. A representative neuron is shown in Fig.1 B. Luciferase expression was found only in association with GnRH-containing neurons demonstrating that luciferase expression does faithfully reflect mGnRH expression. To define further the promoter element necessary to target hypothalamic luciferase expression, transgenic mice were generated using the proximal −1005 bp of the mGnRH promoter fused to the luciferase reporter gene (−1005mGnRH-LUC). As shown in Table I, six separate founder lines bearing the −1005mGnRH-LUC transgene were generated, and three transgenic lines were found to express luciferase in the neonatal brain. In brain homogenates from 2- to 3-day-old pups, luciferase levels were 7044 ± 472 RLU in the lowest luciferase-expressing line and 22,259 ± 5872 in the highest luciferase-expressing line. In founder lines that incorporated, but did not express the luciferase transgene, luciferase activity was 29.3 ± 9.1 RLU in neonatal brain homogenates. As shown in Fig. 1 C, the anatomic pattern of luciferase expression was similar in the three luciferase-expressing lines. Data from two female offspring of founder 1 and four female offspring of founders 2 and 3 are shown. This smaller fragment of the mGnRH promoter, containing the proximal −1005 bp, also targets luciferase expression to the hypothalamus (584 ± 77, 1221 ± 353, and 1244 ± 104), but lower levels were seen. Hypothalamic luciferase levels were 16–36-fold higher in the transgenic mice bearing the −3446mGnRH-LUC transgene (20,148 ± 6093 and 21,263 ± 8214). Although the mice generated with the −1005mGnRH-LUC transgene expressed much lower levels of luciferase, immunocytochemistry studies detected luciferase in GnRH-containing neurons, and a representative neuron is shown in Fig. 1 D. As with the transgenic mice generated with −3446 bp of the mGnRH gene promoter, luciferase expression in the hypothalamus was found only in association with GnRH-containing neurons demonstrating that luciferase expression does faithfully reflect mGnRH expression. Analysis of the −1005mGnRH-LUC mice demonstrates that the proximal −1005 bp of the mGnRH promoter targets luciferase expression to hypothalamic GnRH-containing neurons, suggesting that the mGnRH hypothalamic specific element is located within the proximal −1005 bp of the mGnRH promoter. Interestingly, as shown in Fig. 1 C, offspring from the three luciferase-expressing founder lines of the −1005mGnRH-LUC mice demonstrated very high levels of luciferase activity in their ovarian homogenates (13,641 ± 652, 12,432 ± 1477, and 32,893 ± 5557). Luciferase activity in the other non-neural tissues was similar to background levels, indicating that ovarian luciferase was not an artifact of where the luciferase transgene incorporated into the mouse genome. The high level of ovarian luciferase expression seen in the mice bearing the −1005mGnRH-LUC transgene was somewhat surprising because there was minimal ovarian expression (154 ± 24 and 41 ± 13) found in the transgenic mice bearing the −3446mGnRH-LUC transgene (Fig. 1 A). No significant ovarian luciferase expression was detected in the −3446mGnRH-LUC mice even after additional female mice (n = 22) from both founder lines bearing the −3446mGnRH-LUC transgene were examined (data not shown). The additional female mice ranged from 24 to 296 days in age and included lactating (n = 4), pregnant (n = 3), pre-pubertal (n = 4), and cycling females (n = 11). These findings demonstrate that the proximal −1005 bp of the mGnRH promoter targets luciferase expression to the ovary, as well as the hypothalamus, suggesting that the GnRH ovarian-specific element is also located within the proximal −1005 bp of the mGnRH promoter. Furthermore, removal of the distal mGnRH promoter region, between −3446 bp and −1005 bp, unmasks ovarian luciferase activity in the −1005mGnRH-LUC mice. Our results suggest that the ovary may expresses GnRH at low levels because low levels of luciferase expression were detected in the −3446mGnRH-LUC mice. This finding suggests that DNA sequences in the distal mGnRH promoter, between −3446 bp and −1005 bp, may normally act as an ovarian GnRH repressor element to mediate the repression of ovarian GnRH. To identify the luciferase-expressing cells in the ovary, immunocytochemistry experiments were performed using adult cycling female mice bearing the −1005mGnRH-LUC transgene. Initially, ovarian sections were labeled with antibodies specific for luciferase and GnRH peptides using the staining protocol used for the brain sections. Luciferase immunoreactivity was amplified using biotinylated secondary antibody with streptavidin-conjugated Alexa Fluor 488. GnRH immunoreactivity was visualized using Cy3 fluorescence. In these studies, no immunostaining for GnRH was detected (data not shown). Additionally, immunostaining for luciferase could not be determined because there was a great deal of nonspecific Alexa Fluor 488 fluorescence seen in our control sections, presumably from the endogenous biotin present in the ovary (data not shown). The immunocytochemistry protocol was modified to eliminate the biotinylated secondary antibody. Ovarian sections were obtained from an adult cycling female mouse. Experimental sections were incubated with an anti-luciferase antibody, whereas control sections were incubated with nonspecific rabbit IgG. Immunoreactivity was detected by Cy3 fluorescence. Representative ovarian follicles are shown in Fig. 2. In the mice bearing the −1005mGnRH-LUC transgene, no ovarian staining was found in mice labeled with nonspecific IgG. In sections incubated with the anti-luciferase antibody, fluorescence was detected in the granulosa cells of some ovarian follicles. The fact that only certain follicles within the ovary contained labeled granulosa cells suggests that the luciferase expression seen in these mice is not the result of nonspecific transgene expression. Our findings suggest that increased GnRH promoter activity occurs in the granulosa cells of certain ovarian follicles, either at particular stages of follicular development or within a particular cohort of ovarian follicles. To define further the promoter element necessary to repress ovarian luciferase expression, transgenic mice were generated with the region between −2078 bp and +23 bp of the mGnRH promoter fused to the luciferase reporter gene (−2078mGnRH-LUC). As shown in Table I, Southern blot analysis identified 17 separate founders bearing the −2078mGnRH-LUC transgene, and luciferase expression was detected in neonatal brains from 8 of these founder lines. In brain homogenates from 2-day-old pups, luciferase activity was 1500 ± 104 RLU in the lowest luciferase-expressing line and 12,983 ± 599 RLU in the highest luciferase-expressing line. In neonatal brain homogenates from founder lines that incorporated, but did not express the luciferase transgene, luciferase activity was 25.2 ± 8.7 RLU. The founder line that consis
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