Exploring the determinants of fracture risk among individuals with spinal cord injury.

UNLABELLED: In this cross-sectional study, we found that areal bone mineral density (aBMD) at the knee and specific tibia bone geometry variables are associated with fragility fractures in men and women with chronic spinal cord injury (SCI). INTRODUCTION: Low aBMD of the hip and knee regions have been associated with fractures among individuals with chronic motor complete SCI; however, it is unclear whether these variables can be used to identify those at risk of fracture. In this cross-sectional study, we examined whether BMD and geometry measures are associated with lower extremity fragility fractures in individuals with chronic SCI. METHODS: Adults with chronic [duration of injury ≥ 2 years] traumatic SCI (C1-L1 American Spinal Cord Injury Association Impairment Scale A-D) reported post injury lower extremity fragility fractures. Dual-energy X-ray absorptiometry (DXA) was used to measure aBMD of the hip, distal femur, and proximal tibia regions, while bone geometry at the tibia was assessed using peripheral quantitative computed tomography (pQCT). Logistic regression and univariate analyses were used to identify whether clinical characteristics or bone geometry variables were associated with fractures. RESULTS: Seventy individuals with SCI [mean age (standard deviation [SD]), 48.8 (11.5); 20 females] reported 19 fragility fractures. Individuals without fractures had significantly greater aBMD of the hip and knee regions and indices of bone geometry. Every SD decrease in aBMD of the distal femur and proximal tibia, trabecular volumetric bone mineral density, and polar moment of inertia was associated with fracture prevalence after adjusting for motor complete injury (odds ratio ranged from 3.2 to 6.1). CONCLUSION: Low knee aBMD and suboptimal bone geometry are significantly associated with fractures. Prospective studies are necessary to confirm the bone parameters reported to predict fracture risk in individuals with low bone mass and chronic SCI.

Developing a Model of Care for Healing Pressure Ulcers With Electrical Stimulation Therapy for Persons With Spinal Cord Injury.

Background: Electrical stimulation therapy (EST) has been shown to be an effective therapy for managing pressure ulcers in individuals with spinal cord injury (SCI). However, there is a lack of uptake of this therapy, and it is often not considered as a first-line treatment, particularly in the community. Objective: To develop a pressure ulcer model of care that is adapted to the local context by understanding the perceived barriers and facilitators to implementing EST, and to describe key initial phases of the implementation process. Method: Guided by the Knowledge-to-Action (KTA) and National Implementation Research Network (NIRN) frameworks, a community-based participatory research (CBPR) approach was used to complete key initial implementation processes including (a) defining the practice, (b) identifying the barriers and facilitators to EST implementation and organizing them into implementation drivers, and (c) developing a model of care that is adapted to the local environment. Results: A model of care for healing pressure ulcers with EST was developed for the local environment while taking into account key implementation barriers including lack of interdisciplinary collaboration and communication amongst providers between and across settings, inadequate training and education, and lack of resources, such as funding, time, and staff. Conclusions: Using established implementation science frameworks with structured planning and engaging local stakeholders are important exploratory steps to achieve a successful sustainable best practice implementation project.

Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I.

We proposed that a cell-selective regulatory protein coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. In this report, we identify a 53-kilodalton protein, termed steroidogenic factor 1 (SF-1), that interacts with the related promoter elements from these steroidogenic enzymes, and we isolate and characterize a cDNA that very likely encodes this protein. We first showed that nuclear extracts from bovine adrenal glands interact with the mouse steroidogenic regulatory elements, forming complexes indistinguishable from those produced by nuclear extracts from mouse Y1 adrenocortical cells. These bovine adrenal extracts were subjected to sequential ion exchange and affinity chromatography to yield a highly enriched preparation of SF-1. The predominant protein in the affinity-purified preparation comigrated with shift activity and had a mol wt of 53,000; UV cross-linking experiments demonstrated directly that this 53-kilodalton protein interacted with the steroidogenic regulatory element. Even with this marked enrichment, affinity-purified SF-1 bound six steroidogenic regulatory elements. These results support strongly the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles those of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family. By screening a Y1 cell cDNA library with the DNA-binding region of the H-2RIIBP nuclear hormone receptor cDNA, we isolated a cDNA that is selectively expressed in steroidogenic cells. When expressed as a glutathione S-transferase fusion protein in Escherichia. coli, the protein encoded by this cDNA interacts with all six related steroidogenic regulatory elements with a binding specificity indistinguishable from that of SF-1. Surprisingly, the sequence of the putative DNA-binding domain of this cDNA matches exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor fushi tarazu-factor I. The demonstration that a member of the nuclear hormone receptor family interacts with the steroidogenic regulatory elements provides intriguing insights into possible mechanisms by which these essential genes are regulated.

Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression.

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.

Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues.

In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcription initiation site is located 23 bp 3' of a putative TATA box. When a revised rat sequence was compared to that of the human aromatase PII promoter by BEST-FIT analysis, a region of about 300 bp was identified that was 80% conserved between the two promoters. A potential SF-1 site, CCAAGGTCA, was identified at position -82 within this region. An oligonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific complex was observed with nuclear extracts from gonadal steroidogenic tissues but was absent with nuclear extracts from nonsteroidogenic tissues. The role of SF-1 in this steroidogenic cell-specific complex was next addressed more directly. Bacterial extracts containing an SF-1-glutathione S-transferase fusion protein interacted specifically with the putative SF-1 site, and polyclonal antisera against SF-1-glutathione S-transferase specifically abolished the complex formed with nuclear extracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the aromatase SF-1 element increased expression of an SV40 promoter/luciferase construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in the regulation of steroid hydroxylase gene expression in nonadrenal tissues, significantly extending previous studies in adrenocortical cells.

Steroidogenic factor-1 binding and transcriptional activity of the cholesterol side-chain cleavage promoter in rat granulosa cells.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.

Cytomegalovirus ventriculoencephalitis presenting as a Wernicke's encephalopathy-like syndrome.

Cytomegalovirus ventriculoencephalitis (CMV-VE) is a devastating opportunistic infection seen most frequently in patients with AIDS. The authors describe eight patients with AIDS and CMV-VE, who developed the clinical features of the Wernicke-Korsakoff syndrome, including impaired memory, confabulation, nystagmus, ophthalmoplegia, and ataxia. CMV-VE is perhaps a more frequent cause of the Wernicke-Korsakoff syndrome than traditional associations.

A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.

Activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching.

Cultured bovine adrenocortical cells reach replicative senescence after 100-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17+ to CYP17-, where '+' and '-' refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17 alpha-hydroxylase). We used luciferase reporter constructs to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing -2544 to +29 and -488 to +29 of the 5' region of CYP17 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the CYP17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidogenic factor 1 (SF-1) is essential for endocrine development and function.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, initially was isolated as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Thereafter, analyses of sites of SF-1 expression during mouse embryological development hinted at considerably expanded roles for SF-1, roles that were strikingly confirmed through the analyses of SF-1 knockout mice. These SF-1 knockout mice exhibited adrenal and gonadal agenesis, associated with male-to-female sex reversal of their internal and external genitalia and death from adrenocortical insufficiency. These findings showed unequivocally that SF-1 is essential for the embryonic survival of the primary steroidogenic organs. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), establishing that SF-1 regulates reproductive function at all three levels of the hypothalamic-pituitary gonadal axis. This article reviews the experiments that have defined these essential roles of SF-1 in endocrine development and highlights important areas for future studies.

Demethylation of specific sites in the 5'-flanking region of the CYP17 genes when bovine adrenocortical cells are placed in culture.

DNA methylation of CYP17 (steroid 17 alpha-hydroxylase) was studied in bovine adrenocortical cells, which lose the capacity to express this tissue-specific gene in culture by phenotypic switching. Restriction enzyme digestions, and sequencing of a lambda clone of a second CYP17 gene (CYP17A2), showed that there are at least three CYP17 genes in the bovine genome. Southern blotting of DNA digested with Msp I or Hpa II together with Eco RI was used to investigate the methylation status of Hpa II sites at -1.0 kb (H1), -1.8 kb (H2), and -2.3 kb (H4) in CYP17A1 and CYP17A2 and at -0.7 kb (H0) in CYP17A3. In cells and tissues other than white blood cells, H0 was nonmethylated whereas H1 was always methylated; H2 and H4 showed variation in methylation status among different cells and tissues. In particular, whereas H4 was methylated in the bovine adrenal cortex in vivo, there was a rapid and complete demethylation at H4 when adrenocortical cells were placed in culture. Sites downstream from H4 did not change methylation over the first six passages in culture; additionally, the coding region of CYP17 remained fully methylated under all conditions. In contrast to adrenocortical cells, DNA from fibroblasts was nonmethylated at H2, whereas all downstream sites were fully methylated. Digestion with another methylation-sensitive enzyme, Bsa HI, which has a site between H2 and H4, showed that this region is methylated in intact adrenal cortex but nonmethylated both in cultured adrenocortical cells and in fibroblasts. The specific changes in methylation at this site and at H4 in adrenocortical cells indicate a reproducible, environmentally determined change in methylation in adrenocortical cells when they are placed in culture.

A cell-specific nuclear receptor regulates the steroid hydroxylases.

Recent studies of the gene regulation of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1). SF-1 binds to shared promoter elements upstream of the steroid hydroxylases to mediate their coordinate expression in steroidogenic cells. Analyses of SF-1 expression during mouse embryonic development showed that SF-1 is expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting an intimate link between SF-1 and steroidogenic cell differentiation. Finally, in gene disruption experiments, the gene encoding SF-1 was shown to be essential for development of the adrenal glands and gonads. These results establish the essential role of this orphan nuclear receptor in the development and function of the primary steroidogenic tissues.

Benzocaine-induced methemoglobinemia.

Methemoglobinemia is an uncommon but important complication associated with the use of topical anesthetics. We describe four cases of methemoglobinemia induced by topical benzocaine use. We review pathophysiology, early diagnosis, and therapy for this reversible yet potentially fatal condition. Physicians who use procedures involving the application of topical anesthetics need to be aware of this side effect to prevent significant morbidity and mortality.

Degradation of orcinol by Aspergillus niger.

Aspergillus niger could utilize orcinol (5-methyl-resorcinol or 3,5-dihydroxytoluene) as the sole source of carbon and energy. In the first step of catabolism A. niger hydroxylates orcinol to form 2,3,5-trihydroxytoluene. Its oxidized form, 2-hydroxy-6-methyl-1,4-benzoquinone, was also formed in the culture medium during growth of this organism. Orcinol-grown cells showed a net increase in the intracellular acetate pool, compared with glucose-grown cells. Cell-free extracts of orcinol-grown cells showed higher activity of orcinol hydroxylase, catechol 1,2-oxygenase, and isocitrate lyase than that of glucose-grown cells. Both orcinol-grown and resorcinol-grown cells exhibit similar respiratory activity on all the substrates checked.

Function of steroidogenic factor 1 (SF1) ligand-binding domain in gene activation and interaction with AP1.

The nuclear receptor SF1 is an essential mediator in ventromedial hypothalamus-pituitary-gonadal development. As with other nuclear receptors, SF1 possesses a DNA-binding domain composed of two zinc fingers and a ligand-binding domain containing a ligand-dependent activation sequence termed AF2. To dissect the domain function of SF1, we examined various SF1 mutants in mouse adrenocortical Y1 cells and human placental JEG3 cells. Destruction of the AF2 structure removed 73-90% transactivation activity, suggesting that AF2 is indispensable for transactivation. Mutants carrying the DNA-binding domain but lacking the AF2 or the ligand-binding domain blocked the activity of normal SF1. Disrupting the zinc finger diminished the dominant negative effect of mutant. Cotransfection of SF1 with AP1 showed that the two transcription factors cooperated to activate gene expression. Some mutants lost the synergistic action with AP1, while some retained partial activity. These experiments delineate the functional domains of SF1.

Changes in gene expression during senescence of adrenocortical cells in culture.

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B >CYP21 >CYP17 >CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at -2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.

Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols.

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo.

A cell-specific nuclear receptor plays essential roles in adrenal and gonadal development.

Recent analyses of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1), in their coordinate, cell-selective expression. SF-1 was proposed to regulate the steroid hydroxylases by interacting with shared promoter elements in their 5'-flanking regions. During mouse embryonic development, SF-1 was expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting a key role in steroidogenic cell differentiation. Finally, disruption of the gene encoding SF-1 revealed its essential function in the development of the adrenal glands and gonads and in pituitary gonadotrope function. These studies suggest that SF-1 acts at multiple levels of the reproductive axis to maintain reproductive competence.

The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.