Search

The role of IGF-I in the negative regulation of GH expression and release is demonstrated by in vitro and in vivo models; however, the targets and mechanisms of IGF-I remain unclear. We have developed a cell-specific knockout mouse in which the IGF-I receptor was ablated from the somatotroph in order to validate and characterize IGF-I negative regulation; we termed this the somatotroph IGF-I receptor knockout (SIGFRKO) mouse. The SIGFRKO mice demonstrated increased GH gene expression and secretion as well as increased serum IGF-I. Compensatory changes were noted with decreased GHRH and increased somatostatin mRNA expression levels. SIGFRKO mice had normal linear growth, but by 14 wk of age weighed significantly less than controls. Furthermore, metabolic studies revealed SIGFRKO mice had significantly less fat mass and body percent fat. These data support somatotroph IGF-I negative regulation and suggest that hypothalamic feedback limits the extent of GH release. The SIGFRKO mouse is a model delineating the mechanisms of IGF-I regulation in the hypothalamic-pituitary axis and demonstrates compensatory mechanisms that mediate growth and metabolic function in mammals.

Hypopituitarism is defined as the deficiency of one or more of the hormones secreted by the pituitary gland. Several developmental factors necessary for pituitary embryogenesis and hormone secretion have been described, and mutations of these genes in humans provide a molecular understanding of hypopituitarism. Genetic studies of affected patients and their families provide insights into possible mechanisms of abnormal pituitary development; however, mutations are rare. This review characterizes several of these developmental proteins and their role in the pathogenesis of hypopituitarism. Continuing research is required to better understand the complexities and interplay between these pituitary factors and to make improvements in genetic diagnosis that can lead to early detection and provide a future cure.

Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1-7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10(-9)M kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4h of treatment in both cell lines. Negative regulation by 17beta-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17beta-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin-GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1-7 and GN11 cell lines.

Mice lacking estrogen receptor alpha in the pituitary gonadotroph (PitEsr1KO) were generated to determine the physiologic role of pituitary estrogen signaling in the reproductive axis. PitEsr1KO female mice are subfertile or infertile and have elevated levels of serum luteinizing hormone (LH) and LH beta subunit gene expression, reflecting a lack of estrogen negative feedback effect on the gonadotroph. While serum LH values are elevated in PitEsr1KO mice, the degree of elevation is much less than that observed in ESR1-null mice, indicating that the hypothalamus must also have an important role in estrogen negative feedback. PitEsr1KO mice also demonstrate a defect in estrogen positive feedback, as surge LH values and estrous cyclicity are absent in these mice. Although sex steroid feedback in the reproductive axis is thought to involve discrete anatomic regions that mediate either a positive or negative estrogen effect, PitEsr1KO mice demonstrate novel evidence that localizes both estrogen positive feedback and estrogen negative feedback to the gonadotroph, which suggests that they may be mechanistically related.

Insulin resistance and elevated glucagon levels result in nonsuppressible hepatic glucose production and hyperglycemia in patients with type 2 diabetes. The CREB coactivator complex controls transcription of hepatic gluconeogenic enzyme genes. Here, we show that both the antidiabetic agent metformin and insulin phosphorylate the transcriptional coactivator CREB binding protein (CBP) at serine 436 via PKC iota/lambda. This event triggers the dissociation of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme gene expression. Mice carrying a germline mutation of this CBP phosphorylation site (S436A) demonstrate resistance to the hypoglycemic effect of both insulin and metformin. Obese, hyperglycemic mice display hepatic insulin resistance, but metformin is still effective in treating the hyperglycemia of these mice since it stimulates CBP phosphorylation by bypassing the block in insulin signaling. Our findings point to CBP phosphorylation at Ser436 by metformin as critical for its therapeutic effect, and as a potential target for pharmaceutical intervention.

Estrogen plays an essential role in the regulation of the female reproductive hormone axis, and specifically is a major regulator of GnRH neuronal function in the female brain. GnRH neuronal cell lines were used to explore the direct effects of estradiol on gene expression in GnRH neurons. The presence of estrogen receptor (ER) binding sites was established by a receptor-binding assay, and estrogen receptor alpha and beta mRNA were identified in GN11 cells and ERbeta in GT1-7 cells using RT-PCR analysis of mRNA. ERalpha was more abundantly expressed in GN11 cells than ERbeta as assessed by real-time PCR. Additionally, GN11 cells expressed significantly more of both ERalpha and beta than GT1-7 cells. Functional studies in GN11 and GT1-7 demonstrated estrogen down regulation of endogenous mouse GnRH mRNA levels using quantitative real-time PCR (qRT-PCR). Correspondingly, estradiol also reduced secretion of GnRH from both the GN11 and GT1-7 cell lines. Since estradiol has been shown to regulate progesterone receptor (PR) expression; similar studies were performed demonstrating an estradiol mediated increase in PR in both cell lines. Estradiol regulation of ER expression was also explored and these studies indicated that estradiol decreased ERalpha and ERbeta mRNA levels in a dose-dependent manner in GN11 and GT1-7 cells. These effects were blocked by the addition of the estrogen receptor antagonist ICI 182,780. Both PPT, a specific ERalpha agonist, and DPN, a specific ERbeta agonist, inhibited GnRH gene expression in GN11 cells, but only DPN inhibited GnRH gene expression in GT1-7 cells, consistent with their undetectable levels of ERalpha expression. These studies characterize a direct inhibitory effect of estradiol on GnRH in GnRH neurons, and a direct stimulatory effect of estradiol on PR gene expression. In addition, the agonist studies indicate that there is a functional overlap of ERalpha and ERbeta regulation in GnRH neurons. These studies may give insight into the molecular regulation of estrogen negative feedback in the central reproductive axis.

Purpose of Review: Puberty is the developmental process that culminates in reproductive capability. It is initiated by the release of gonadotropin-releasing hormone from specialized neurons of the hypothalamus to stimulate hormonal cascades and gonadal activation. The age of pubertal onset in girls may be younger than in previous decades, emphasizing the gaps in knowledge about pubertal onset and factors that modulate it. This review describes the state of the debate on the age of pubertal initiation, recent insights into the physiology of female puberty and its relationship to childhood obesity, and the regulation of gonadotropin-releasing hormone neurons at puberty., Recent Findings: Recent studies suggest that the average age of pubertal onset is decreasing in American girls, sparking controversy in defining the age at which puberty is considered precocious. Obese girls have hyperinsulinemia and hyperandrogenemia prepubertally, but it is unclear whether these factors play a role in the early onset of puberty in obese girls. The kisspeptin/G protein-coupled receptor 54 pathway is important for pubertal initiation; an activating mutation in the pathway has been associated with precocious puberty., Summary: The recent trend toward obesity has been proposed to play a role in the cause of early puberty in girls. The molecular mechanisms that initiate puberty are slowly being elucidated, with the discovery of kisspeptin prompting a novel direction in reproductive research.

The estrogen receptor-alpha knockout (ERalphaKO, ERalpha-/-) mice were generated via the Cre-loxP system by mating floxed ERalpha mice with beta-actin (ACTB)-Cre mice. The impact of ERalpha gene deletion in the male reproductive system was investigated. The ACTB-Cre/ERalpha(-/-) male mice are infertile and have lost 90% of epididymal sperm when compared with wild-type mice. Serum testosterone levels in ACTB-Cre/ERalpha(-/-) male mice are 2-fold elevated. The ACTB-Cre/ERalpha(-/-) testes consist of atrophic and degenerating seminiferous tubules with less cellularity in the disorganized seminiferous epithelia. Furthermore, the ventral and dorsal-lateral prostates of ACTB-Cre/ERalpha(-/-) mice display reduced branching morphogenesis. Loss of ERalpha could also be responsible for the decreased fibroblast proliferation and changes in the stromal content. In addition, we found bone morphogenetic protein, a mesenchymal inhibitor of prostatic branching morphogenesis, is significantly up-regulated in the ACTB-Cre/ERalpha(-/-) prostates. Collectively, these results suggest that ERalpha is required for male fertility, acts through a paracrine mechanism to regulate prostatic branching morphogenesis, and is involved in the proliferation and differentiation of prostatic stromal compartment.

Conventional estrogen receptor alpha knockout (neo-ERalphaKO, neo-ERalpha(-/-)) mice contain a truncated and chimeric ERalpha fusion protein that retains 35% estrogen-dependent transactivation activity, and therefore the in vivo ERalpha function is difficult to study thoroughly. Furthermore, these neo-ERalpha(-/-) mice cannot be used for tissue and temporal specific ERalpha deletion. Therefore, there is a clear need to establish a floxed ERalpha mouse line that can knockout ERalpha specifically and completely in each selected cell type. Here we generated floxed ERalpha mice using a self-excising ACN (tACE-Cre/Neo) cassette. Mating the floxed ERalpha mice with ACTB-Cre mice produces a deletion of the floxed allele disrupting the reading frame of the ERalpha transcript so that no ERalpha protein is detected in the ACTB-Cre/ERalpha(-/-) mice. Expression of ERalpha target genes, such as G-6-PD and lactoferrin, is diminished by over 90% in the ACTB-Cre/ERalpha(-/-) uterus, but not in the neo-ERalpha(-/-) uterus. Furthermore, we also validated that ACTB-Cre/ERalpha(-/-) females have a hypoplastic internal genital tract, polycystic ovaries with hemorrhagic follicles, infertility, and higher body weight. Together, our data clearly demonstrate that the newly established floxed ERalpha mouse is a reliable mouse model for future studies of ERalpha roles in vivo in the selective estrogen target tissues. The complete knockout of ERalpha in the ACTB-Cre/ERalpha(-/-) mice will also provide an improved mouse model to study the role of ERalpha in vivo.

Context: Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies in more than one anterior pituitary hormone. Mutations in developmental factors responsible for pituitary cell specification and gene expression have been found in CPHD patients. OTX2, a bicoid class homeodomain protein, is necessary for both forebrain development and transactivation of the HESX1 promoter, but as of yet, has not been associated with CPHD., Objective: The goal of this study was to identify and characterize novel mutations in pituitary specific transcription factors from CPHD patients., Design: Genomic DNA was isolated from patients with hypopituitarism to amplify and sequence eight pituitary specific transcription factors (HESX1, LHX3, LHX4, OTX2, PITX2, POU1F1, PROP1, and SIX6). Characterization of novel mutations is based on structural and functional studies., Results: We describe two unrelated children with CPHD who presented with neonatal hypoglycemia, and deficiencies of GH, TSH, LH, FSH, and ACTH. Magnetic resonance imaging revealed anterior pituitary hypoplasia with an ectopic posterior pituitary. A novel heterozygous OTX2 mutation (N233S) was identified. Wild-type and mutant OTX2 proteins bind equivalently to bicoid binding sites, whereas mutant OTX2 revealed decreased transactivation., Conclusions: A novel mutation in OTX2 binds normally to target genes and acts as a dominant negative inhibitor of HESX1 gene expression. This suggests that the expression of HESX1, required for spaciotemporal development of anterior pituitary cell types, when disrupted, results in an absent or underdeveloped anterior pituitary with diminished hormonal expression. These results demonstrate a novel mechanism for CPHD and extend our knowledge of the spectrum of gene mutations causing CPHD.

Objective: To assess weight-related beliefs and concerns of overweight urban, African-American children, their parents, and community leaders before developing a family-based intervention to reduce childhood overweight and diabetes risk., Design: We conducted 13 focus groups with overweight children and their parents and eight semistructured interviews with community leaders., Participants and Setting: Focus group participants (N = 67) from Chicago's South Side were recruited through flyers in community sites. Interview participants (N = 9) were recruited to sample perspectives from health, fitness, education, civics, and faith leaders., Results: Community leaders felt awareness was higher for acute health conditions than for obesity. Parents were concerned about their children's health, but felt stressed by competing priorities and constrained by lack of knowledge, parenting skills, time, and financial resources. Parents defined overweight in functional terms, whereas children relied upon physical appearances. Children perceived negative social consequences of overweight. Parents and children expressed interest in family-based interventions to improve nutrition and physical activity and offered suggestions for making programs interesting., Conclusions: This study provides insights into the perspectives of urban, African-American overweight children, their parents, and community leaders regarding nutrition and physical activity. The specific beliefs of these respondents can become potential leverage points in interventions.

Puberty is the developmental process that culminates in reproductive capability and is the result of a complex series of molecular and physiological events. The release of gonadotropin-releasing hormone from specialized neurons of the hypothalamus begins the hormonal cascade that causes gonadal activation and the physical changes of puberty. Several factors have been proposed to influence the activation of the hypothalamus to trigger puberty, but the involved pathways have not been fully elucidated. The recent observations that the age of pubertal onset may be lowering in American girls calls attention to the lack of knowledge of modulating factors that affect the pubertal process. Genes necessary for puberty have been found by studying persons who do not achieve puberty; such studies have provided insights into the pathways necessary for pubertal development. A multidisciplinary focus is required to elucidate the complex mechanisms involved in the initiation and progression of puberty.

Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line--an immortalized GnRH-secreting neuronal cell line--insulin maximally increases Egr-1 mRNA after 30min of treatment and Egr-1 protein and GnRH mRNA after 60min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between -67 and -76bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.

The observation that some adults with childhood-onset GH deficiency have low bone mineral density, low lean body mass, diminished quality of life, abnormal lipids, and impaired cardiac function, all of which may improve after treatment with GH, has prompted pediatric endocrinologists to reevaluate the practice of discontinuing GH in all patients after attainment of final adult height. The treatment of adolescents to prevent the metabolic complications of GH deficiency is an emerging practice. Studies addressing the evaluation and care of adolescents during this period and the benefits of GH in this setting are conflicting. Our approach in determining which adolescents to retest, when and how to test for persistent GH deficiency, and which subjects to treat is discussed in the context of available clinical data.

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.

Inducing tissue-specific genetic alterations under temporal control allows for the analysis of gene function in particular cell types at specified points in time. We have generated a system for tetracycline-controlled expression of Cre recombinase in mice using the unique CreTeR vector. The gonadotroph-specific bovine alpha-subunit (Balpha) promoter fragment was subcloned into the CreTeR vector, creating a technique for highly regulated expression of Cre recombinase exclusively in pituitary gonadotrophs. Control of Cre recombinase in the CreTeR vector was demonstrated in LbetaT2 pituitary cell lines, where Cre protein was detected in cells treated with doxycycline, but not in untreated cells. In transgenic mice, Cre was expressed in pituitary gonadotrophs of mice treated with doxycycline, but not in non-pituitary tissues or in transgenic mice not treated with doxycycline. We demonstrated Cre expression in the gonadotroph by immunostaining showing co-localization of Cre recombinase with the beta-subunit of LH (LH-beta). Furthermore, by crossing Balpha/CreTeR with R26R mice, we were able to demonstrate functional recombination within pituitary gonadotrophs, detected by lacZ expression. The Balpha/CreTeR mice described here can be used to study the function of virtually any gene in the gonadotroph; in particular, this will be useful in studying genes, which may have distinct roles in development and in the adult.

Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHbeta gene expression by GnRH and insulin was examined in LbetaT2 cells. Endogenous LHbeta mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences -140 to +1 in the mouse LHbeta gene. Insulin together with GnRH stimulates activity of an LHbeta-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences -51 to -42 in the LHbeta gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHbeta DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.

Context: Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA-binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA., Objective: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding., Design: DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W., Results: In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes., Conclusions: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.

The science of measuring bone mineral density has developed rapidly and, with it, an improved understanding of the efficacy and safety of various therapeutic interventions in adults. In contrast, the meaning and precision of such measurements in children are equivocal, and the concept of treatment for low bone density in the young patient is still largely undecided. In this report we review the present state of knowledge regarding the use of bisphosphonates during childhood to ameliorate the skeletal abnormalities associated with osteogenesis imperfecta, idiopathic juvenile osteoporosis, fibrous dysplasia of bone and cerebral palsy. Because of the paucity of long-term studies among children regarding the safety and efficacy of these drugs, it is difficult to formulate strong evidence-based recommendations for their use, except perhaps in children with osteogenesis imperfecta.

HESX1 is a paired-like homeodomain transcription factor that functions as a repressor of PROP1-mediated gene stimulation. Mutations in HESX1 have been implicated in cases of septooptic dysplasia and congenital hypopituitarism. All mutations in HESX1 identified to date have resulted in impaired DNA binding and defective HESX1 action. We have identified a novel HESX1 mutation in genomic nucleotide position 1684 (g.1684delG), which results in a mutant protein with increased DNA binding. In turn, this mutation causes increased repression of PROP1-dependent gene activity. These data suggest that enhancement of transcriptional repression during pituitary organogenesis is a novel mechanism for the development of congenital pituitary disorders.

Pituitary gland commitment from oral ectoderm occurs in response to inductive signals from the neuroepithelium of the ventral diencephalon. Invagination of the oral ectoderm leads to the creation of Rathke's pouch. Intensified cell proliferation within Rathke's pouch results in formation of the anterior pituitary lobe. Subsequently, highly differentiated cell types arise sequentially due to overlapping, but distinct, spatial and temporal patterns of signaling molecules and transcription factors. Mutations in some of the pituitary-specific transcription factors have been identified in patients with hypopituitarism, confirming the role of these factors in pituitary development.

The human GnRH (hGnRH) gene is expressed, and the GnRH decapeptide produced, primarily in the GnRH neurons of the diencephalon. The molecular elements important for the cell-specific expression and regulation of the hGnRH gene are not well established at this time; therefore, we have used a transgenic mouse model to isolate cis-regulatory elements important for directing gene expression to GnRH neurons in the hypothalamus. Gene constructs consisting of various promoter deletion fragments of the hGnRH gene fused to the luciferase (LUC) reporter gene have been used to create transgenic mouse lines. Cell-specific expression, with the criterion being luciferase expression directed to GnRH neurons of the hypothalamus, was observed when 992 bp, but not 795 bp, of the hGnRH gene promoter were used. Tissue-specific expression was also observed when a deletion construct containing the region from -992 to -763 was fused to a minimal 48-bp promoter fragment fused to LUC. These data indicate that the region between -992 and -795 contains elements both essential and sufficient for targeting gene expression to GnRH neurons. This promoter region was found to contain two DNA-binding sites for the POU class of transcription factors, each of which specifically interacted with the POU homeodomain proteins Brn-2 and Oct-1. Functional studies demonstrated that Brn-2 increased promoter activity of the human and mouse GnRH genes.

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 tissues in 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.