Search

Robert Hromas,1 Robert Leverence,1 Lazarus K Mramba,2 J Larry Jameson,3 Caryn Lerman,3 Thomas L Schwenk,4 Ellen M Zimmermann,2 Michael L Good51The Office of the Dean, Department of Medicine, University of Texas Health Science Center San Antonio, San Antonio, TX, USA; 2Department of Medicine, College of Medicine, University of Florida Health, Gainesville, FL, USA; 3Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 4Department of Family Medicine, School of Medicine, University of Nevada Reno, Reno, NV, USA; 5Department of Anesthesiology, College of Medicine, University of Florida Health, Gainesville, FL, USAAbstract: Economic pressure has led the evolution of the role of the medical school dean from a clinician educator to a health care system executive. In addition, other dynamic requirements also have likely led to changes in their leadership characteristics. The most important relationship a dean has is with the chairs, yet in the context of the dean’s changing role, little attention has been paid to this relationship. To frame this discussion, we asked medical school chairs what characteristics of a dean’s leadership were most beneficial. We distributed a 26-question survey to 885 clinical and basic science chairs at 41 medical schools. These chairs were confidentially surveyed on their views of six leadership areas: evaluation, barriers to productivity, communication, accountability, crisis management, and organizational values. Of the 491 chairs who responded (response rate =55%), 88% thought that their dean was effective at leading the organization, and 89% enjoyed working with their dean. Chairs indicated that the most important area of expertise of a dean is to define a strategic vision, and the most important value for a dean is integrity between words and deeds. Explaining the reasons behind decisions, providing good feedback, admitting errors, open discussion of complex or awkward topics, and skill in improving relations with the teaching hospital were judged as desirable attributes of a dean. Interestingly, only 23% of chairs want to be a dean in the future. Financial acumen was the least important skill a chair thought a dean should hold, which is in contrast to the skill set for which many deans are hired and evaluated. After reviewing the literature and analyzing these responses, we assert that medical school chairs want their dean to maintain more traditional leadership than that needed by a health care system executive, such as articulating a vision for the future and keeping their promises. Thus, there appears to be a mismatch between what medical school chairs perceive they need from their dean and how the success of a dean is evaluated. Keywords: academic medicine deans, leadership characteristics, organizational values

Purpose: The murder of George Floyd in 2020 prompted a national demand for cultural transformation to confront the systemic racism prevalent in the country. Academic medical centers were not exempt from this urgent call. This article evaluates the efficacy of a strategic process in fostering cultural transformation within an academic medical system., Methods: A whole-scale strategic planning process was implemented over 13 months, involving multiple working groups representing key stakeholders from each entity across the system, an anonymous survey, a communication plan, and a balanced scorecard to monitor progress. More than 5500 voices, 160 recommendations, 122 data gathering sessions, and town hall meetings contributed to the creation and implementation of vital action items and a strategic framework. The Diversity Engagement Survey (DES) was administered 18 months following the process launch., Results: Of the 45,554 employees, students, faculty, and trainees, 96.5% completed unconscious bias education within the fiscal year and 76% of action items, termed "Just Do Its," were completed. Mission, vision, values, and strategic priorities were crafted to serve as a framework for intermediate and long-term actions. The DES revealed improvement in the "respect" attribute of an inclusive culture, and 64% of respondents confirmed that action for cultural transformation is addressing racism both within and outside of the institution., Conclusion: Implementing a shared purpose, engaging multiple working groups representing key stakeholders, and empowerment of stakeholders to implement changes, in conjunction with the development of a strategic framework addressing structural racism, resulted in the completion of vital action items to initiate cultural change., Competing Interests: The authors declare no conflicts of interest relevant to this article., (© Eve J. Higginbotham et al., 2023; Published by Mary Ann Liebert, Inc.)

Academic medical center (AMC) faculty, administrators, and leaders have the critical tasks of teaching and training the next generation of health care providers and biomedical researchers, as well as generating new knowledge that improves the health of all. In the United States, medical schools and their affiliated hospitals train remarkably high-quality physicians and scientists, and the research conducted at these institutions results in advances in health. To that end, AMCs have become essential engines for driving better health in the United States and the rest of the world; they also have become essential engines driving the economies of their respective communities and regions. The education and research missions, however, require subsidization because tuition and extramural grant funding do not cover the costs of these endeavors. This subsidization largely has come from revenues generated by AMCs' clinical endeavors. The viability of this cross-subsidization, however, is increasingly threatened in the current clinical environment. The authors of this Perspective discuss these issues in depth and provide some concrete recommendations to address these challenges. They hope to stimulate discussion and, ultimately, ensure the financial viability of U.S. AMCs-a national resource of utmost importance. Recommendations to sustain research include creating strategic biomedical research plans, developing a defined and sustained model to support National Institutes of Health funding that keeps pace with inflation, and evolving funding mechanisms. Recommendations to sustain medical education include limiting student debt, creating more cost-effective curricula, and ensuring that clinical training opportunities that meet national standards are available to students.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine., (Copyright © 2015, American Association for the Advancement of Science.)

Background: The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by xenobiotic substances such as dioxin. After activation, it binds to dioxin response elements of DNA, thereby inducing transcription of a variety of xenobiotic metabolizing enzymes. To investigate whether AhR-activating substances accumulate in patients with endocrine disorders, we tested serum samples for AhR-stimulating activity., Methods: Serum AhR-stimulating activity was evaluated by exposing the HepG2 cells transiently transfected with an AhR-responsive reporter plasmid to serum samples. On the basis of preliminary findings that implicated methimazole (MMI), wild-type and AhR-null mice were treated with MMI, and their plasma AhR-stimulating activities and thyroxine levels were quantified., Results: In 28 randomly chosen patients, 7 out of 10 Graves' disease patients exhibited increased serum AhR-stimulating activity. The increased activity did not correlate with thyroid hormone status. However, we hypothesized that it might be caused by MMI. Subsequent analyses revealed that in 25 of 26 MMI-treated Graves' patients, serum samples collected after the MMI treatment had significantly higher AhR-stimulating activity compared to samples obtained when the same patients were not on MMI. By contrast, serum AhR-stimulating activity was unchanged in samples from the seven patients on propylthiouracil (PTU) compared to serum taken before the PTU treatment. In vitro experiments demonstrated that an MMI metabolite 3-methyl-2-thiohydantoin, but not MMI, activated AhR. MMI increased plasma AhR-stimulating activities and reduced plasma thyroxine concentrations, in both wild-type and AhR-deficient mice., Conclusions: Graves' patients taking MMI have increased serum AhR-stimulating activity, which is unrelated to thyroid hormone status, but correlates with MMI treatment. The AhR activation is likely caused by 3-methyl-2-thiohydantoin. Further studies are required to determine the potency of 3-methyl-2-thiohydantoin as an AhR activator and the significance of the differences between MMI and PTU observed in this study.

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; also known as NROB1, nuclear receptor subfamily 0, group B, member 1) encodes a nuclear receptor that is expressed in embryonic stem (ES) cells, steroidogenic tissues (gonads, adrenals), the ventromedial hypothalamus (VMH), and pituitary gonadotropes. Humans with DAX1 mutations develop an X-linked syndrome referred to as adrenal hypoplasia congenita (AHC). These boys typically present in infancy with adrenal failure but later fail to undergo puberty because of hypogonadotropic hypogonadism (HHG). The adrenal failure reflects a developmental abnormality in the transition of the fetal to adult zone, resulting in glucocorticoid and mineralocorticoid deficiency. The etiology of HHG involves a combined and variable deficiency of hypothalamic GnRH secretion and/or pituitary responsiveness to GnRH resulting in low LH, FSH and testosterone. Treatment with exogenous gonadotropins generally does not induce spermatogenesis. Animal models indicate that DAX1 also plays a critical role in testis development and function. As a nuclear receptor, DAX1 has been shown to function as a transcriptional repressor, particularly of pathways regulated by other nuclear receptors, such as steroidogenic factor 1 (SF1). In addition to reproductive tissues, DAX1 is also expressed at high levels in ES cells and plays a role in the maintenance of pluripotentiality. Here we review the clinical manifestations associated with DAX1 mutations as well as the evolving information about its function based on animal models and in vitro studies., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4(I242N/I242N) mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4(+/I242N) mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis.

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

The X-linked Sox3 gene encodes a member of the Sry high-mobility group box proteins, which play a role in many developmental processes including neurogenesis and testis development. This study further examined the role of Sox3 in spermatogenesis. Males without Sox3 expression exhibited a similar number of germ cell nuclear antigen-positive germ cells at 1, 5, and 10 d postpartum (dpp) compared to their wild-type littermates, but there was significant germ cell depletion by 20 dpp. However, spermatogenesis later resumed and postmeiotic germ cells were observed by 56 dpp. The VasaCre transgene was used to generate a germ cell-specific deletion of Sox3. The phenotype of the germ cell-specific Sox3 knockout was similar to the ubiquitous knockout, indicating an intrinsic role for Sox3 in germ cells. The residual germ cells in 20 dpp Sox3(-/Y) males were spermatogonia as indicated by their expression of neurogenin3 but not synaptonemal complex protein 3, which is expressed within cells undergoing meiosis. RNA expression analyses corroborated the histological analyses and revealed a gradual transition from relatively increased expression of spermatogonia genes at 20 dpp to near normal expression of genes characteristic of undifferentiated and meiotic germ cells by 84 dpp. Fluorescent-activated cell sorting of undifferentiated (ret tyrosine kinase receptor positive) and differentiated (kit receptor tyrosine kinase-positive) spermatogonia revealed depletion of differentiated spermatogonia in Sox3(-/Y) tubules. These results indicate that Sox3 functions in an intrinsic manner to promote differentiation of spermatogonia in prepubertal mice but it is not required for ongoing spermatogenesis in adults. The Sox3(-/Y) males provide a unique model for studying the mechanism of germ cell differentiation in prepubertal testes.

In addition to its role in reproduction, estradiol-17β is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα-/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα-/- mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17β on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women.

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.

The study objective was to determine whether stromal and/or epithelial estrogen receptor-alpha (ERalpha) is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Four types of tissue recombinants were prepared with cervical stroma (St) and epithelium (Ep) from wild-type (wt) and ERalpha knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing 17beta-estradiol (treatment d 1). Animals were injected sc with relaxin or vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before tissue recombinants were collected at 1000 h on d 10. Relaxin promoted marked proliferation of both epithelial and stromal cells in tissue recombinants containing wt St (P < 0.001) but far lower proliferation in recombinants prepared with ko St, regardless of whether Ep was derived from wt or ko mice. An additional experiment using mice expressing wt ERalpha, a mutant of ERalpha that selectively lacks classical signaling through estrogen response element binding, or no ERalpha demonstrated that ERalpha must bind to an estrogen response element to enable relaxin's proliferative effects. In conclusion, this study shows that ERalpha-expressing cells in St, using a classical signaling pathway, are necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix.

Objective: Mutations of the gsp oncogene are responsible for 30-40% of GH-producing pituitary adenomas and 10% of nonfunctioning pituitary adenomas (NFPAs). However, the pathogenetic mechanism of the remaining pituitary tumours still remains to be identified. Recently, the interaction between the chemokine stromal cell-derived factor 1 and its receptor CXCR4 was found to play an important role in GH production and cell proliferation in various pituitary adenoma cell lines. As CXCR4 is a Gi-coupled chemokine receptor, its constitutive activating mutations may be involved in pituitary tumour formation by cyclic adenosine monophosphate (cAMP)-independent, ERK-related pathways., Patients and Methods: We investigated whether somatic activating-mutations of CXCR4 might be a possible tumourigenic mechanism for gsp-negative GH-secreting pituitary adenomas and NFPAs. Direct sequencing of polymerase chain reaction-amplified products for coding exons of CXCR4 were performed using genomic deoxyribonucleic acid samples from 37 GH-producing pituitary tumour tissues that were negative for the gsp mutation and 14 CXCR4 expressing NFPAs., Results: Immunohistochemical analyses and double immunofluorescent staining of sectioned paraffin-embedded pituitary tissues revealed that CXCR4 is highly expressed in GH-producing pituitary adenomas and NFPAs. Direct sequencing showed that two synonymous mutations in exon 2 (87 C > T and 414 C > T) were detected in 4 out of 51 pituitary tumours., Conclusion: Our results indicate that an activating mutation of the CXCR4 may not be a common pathogenetic mechanism in GH-producing pituitary tumours and NFPAs.

In vitro models have been used to demonstrate that estrogen receptors (ERs) can regulate estrogen-responsive genes either by directly interacting with estrogen-responsive element (ERE) DNA motifs or by interacting with other transcription factors such as AP1. In this study, we evaluated estrogen (E(2))-dependent uterine gene profiles by microarray in the KIKO mouse, an in vivo knock-in mouse model that lacks the DNA-binding function of ERalpha and is consequently restricted to non-ERE-mediated responses. The 2- or 24-h E(2)-mediated uterine gene responses were distinct in wild-type (WT), KIKO, and alphaERKO genotypes, indicating that unique sets of genes are regulated by ERE and non-ERE pathways. After 2 h E(2) treatment, 38% of the WT transcripts were also regulated in the KIKO, demonstrating that the tethered mechanism does operate in this in vivo model. Surprisingly, 1438 E(2)-regulated transcripts were unique in the KIKO mouse and were not seen in either WT or alphaERKO. Pathway analyses revealed that some canonical pathways, such as the Jak/Stat pathway, were affected in a similar manner by E(2) in WT and KIKO. In other cases, however, the WT and KIKO differed. One example is the Wnt/beta-catenin pathway; this pathway was impacted, but different members of the pathway were regulated by E(2) or were regulated in a different manner, consistent with differences in biological responses. In summary, this study provides a comprehensive analysis of uterine genes regulated by E(2) via ERE and non-ERE pathways.

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-alpha signaling. We show that Foxl2 has no effect on classical ERalpha-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERalpha signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERalpha, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERalpha rather than direct action at AP1 binding sites. First, ERalpha is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERalpha and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERalpha signaling and thus modulation by ERalpha/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERalpha in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERalpha stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERalpha and Foxl2 interact and that Foxl2 selectively suppresses ERalpha-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERalpha and Foxl2 to regulate ovarian development and function.

Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17beta (E(2)), which acts via the estrogen receptor alpha isoform (ER alpha) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E(2) inhibits the expression of Kiss1 mRNA. However, E(2) induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E(2) is unknown. ER alpha signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E(2) on Kiss1 and Dyn expression, we studied the effects of E(2) on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ER alpha signaling pathways. We found that stimulation of Kiss1 expression by E(2) in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E(2) in the Arc involved ERE-independent mechanisms. Thus, distinct ER alpha signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E(2) can act within the same neuron through divergent ER alpha signaling pathways to regulate different neurotransmitter genes.

Nonclassical estrogen receptor alpha (ERalpha) signaling can mediate E(2) negative feedback actions in the reproductive axis; however, downstream pathways conveying these effects remain unclear. These studies tested the hypothesis that p21-activated kinase 1 (PAK1), a serine/threonine kinase rapidly activated by E(2) in nonneural cells, functions as a downstream node for E(2) signaling pathways in cells of the preoptic area, and it may thereby mediate E(2) negative feedback effects. Treatment of ovariectomized (OVX) rats with estradiol benzoate (EB) caused rapid and transient induction of phosphorylated PAK1 immunoreactivity in the medial preoptic nucleus (MPN) but not the arcuate nucleus. To determine whether rapid induction of PAK phosphorylation by E(2) is mediated by nonclassical [estrogen response element (ERE)-independent] ERalpha signaling, we used female ERalpha null (ERalpha(-/-)) mice possessing an ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding DNA and can signal only through membrane-initiated or ERE-independent genotropic pathways (ERalpha(-/AA) mice). After 1-h EB treatment, the number of pPAK1-immunoreactive cells in the MPN was increased in both wild-type (ERalpha(+/+)) and ERalpha(-/AA) mice but was unchanged in ERalpha(-/-) mice. Serum luteinizing hormone (LH) was likewise suppressed within 1 h after EB treatment in ERalpha(+/+) and ERalpha(-/AA) but not ERalpha(-/ -) mice. In OVX rats, 5-min intracerebroventricular infusion of a PAK inhibitor peptide but not control peptide blocked rapid EB suppression of LH secretion. Taken together, our findings implicate PAK1 activation subsequent to nonclassical ERalpha signaling as an important component of the negative feedback actions of E(2) in the brain.

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.

Estrogen receptor alpha (ERalpha) mediates estrogen (E2) actions in the brain and is critical for normal reproductive function and behavior. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also participate in several non-classical pathways, including ERE-independent gene transcription via protein-protein interactions with transcription factors and rapid, non-genotropic pathways. To distinguish between ERE-dependent and ERE-independent mechanisms of E2 action in vivo, we have created ERalpha null mice that possess an ER knock-in mutation (E207A/G208A; "AA"), in which the mutant ERalpha cannot bind to DNA but retains activity in ERE-independent pathways (ERalpha(-/AA) mice). Understanding the molecular mechanisms of ERalpha action will be helpful in developing pharmacological therapies that differentiate between ERE-dependent and ERE-independent processes. This review focuses on how the ERalpha(-/AA) model has contributed to our knowledge of ERalpha signaling mechanisms in estrogen regulation of the reproductive axis and sexual behavior.

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

Background: Loss-of-function in the apoptosis-inducing genes is known to facilitate tumorigenesis. AFX (FOXO4), a member of forkhead transcription factors functions as a tumor suppressor and has 2 isoforms, AFXalpha (505 a.a.) and AFXzeta (450 a.a.). In human cancer cells, we identified an N-terminally deleted form of AFXalpha (alpha198-505), translated from a downstream start and 2 short N-terminal AFX proteins (90, and 101 a.a.) produced by aberrant splicing., Methods and Findings: We investigated the expression and role of these AFX variants. Cell transduction study revealed that short N-terminal AFX proteins were not stable. Though alpha(198-505) protein expression was detected in the cytoplasm and nucleus, alpha(198-505) expressing cells did not show a nucleocytoplasmic shuttling mediated by PI3 kinase signaling. Whereas, we observed this shuttling in cells expressing either AFXalpha or AFXzeta protein. AFXzeta and alpha(198-505) lost the ability to transactivate BCL6 or suppress cyclin D2 gene expression. These variants did not induce cancer cell death whereas AFXalpha resulted in apoptosis. We found that AFXzeta and alpha(198-505) suppress the AFXalpha stimulation of BCL6 promoter in a dose dependent manner, indicating dominant negative activity. These variants also inhibited AFXalpha induction of apoptosis., Conclusions: Loss of function by aberrant splicing and the dominant negative activity of AFX variants may provide a mechanism for enhanced survival of neoplastic cells.

Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the beta-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

Estrogen receptor (ER)-alpha mediates estradiol (E(2)) actions in the male gonads and brain and is critical for normal male reproductive function. In the classical pathway, ERalpha binds to estrogen response elements (EREs) to regulate gene transcription. ERalpha can also regulate gene transcription independently of EREs via protein-protein interactions with transcription factors and additionally signal via rapid, nongenomic pathways originating at the cell membrane. This study assessed the degree to which ERE-independent ERalpha signaling can rescue the disrupted masculine sexual behaviors and elevated serum testosterone (T) levels that have been shown to result from ERalpha gene deletion. We utilized male ERalpha null mice that possess a ER knock-in mutation (E207A/G208A; AA), in which the mutant ERalpha is incapable of binding to DNA and can signal only through ERE-independent pathways (ERalpha(-/AA) mice). We found that sexual behavior, including mounting, is virtually absent in ERalpha(-/-) and ERalpha(-/AA) males, suggesting that ERE-independent signaling is insufficient to maintain any degree of normal sexual behavior in the absence of ERE binding. By contrast, ERE-independent signaling in the ERalpha(-/AA) mouse is sufficient to restore serum T levels to values observed in wild-type males. These data indicate that binding of ERs to EREs mediates most if not all of E(2)'s effects on male sexual behavior, whereas ERE-independent ERalpha signaling may mediate E(2)'s inhibitory effects on T production.

Testicular Leydig cells produce testosterone and provide the hormonal environment required for male virilization and spermatogenesis. In utero, fetal Leydig cells (FLCs) are necessary for the development of the Wolffian duct and male external genitalia. Steroidogenic factor 1 (Sf1) is a transcriptional regulator of hormone biosynthesis genes, thus serving a central role in the Leydig cell. Desert hedgehog (Dhh), a Sertoli cell product, specifies the FLC lineage in the primordial gonad through a paracrine signaling mechanism. Postnatally, FLCs are replaced in the testis by morphologically distinct adult Leydig cells (ALCs). To study a putative interaction between Sf1 and Dhh, we crossed Sf1 heterozygous mutant mice with Dhh homozygous null mice to test the function of these two genes in vivo. All of the compound Sf1(+/-); Dhh(-/-) mutants failed to masculinize and were externally female. However, embryonic gonads contained anastomotic testis cords with Sertoli cells and germ cells, indicating that sex reversal was not attributable to a fate switch of the early gonad. Instead, external feminization was attributable to the absence of differentiated FLCs in XY compound mutant mice. ALCs also failed to develop, suggesting either a dependence of ALCs on the prenatal establishment of Leydig cell precursors or that Sf1 and Dhh are both required for ALC maturation. In summary, this study provides genetic evidence that combinatorial expression of the paracrine factor Dhh and nuclear transcription factor Sf1 is required for Leydig cell development.

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) alpha but not ERbeta knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERalpha. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERalpha signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERalpha (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERalpha-null (-/-) background, we examine the ability of ERE-independent ERalpha signaling pathways to convey estrogen feedback regulation of the female hypothalamic-pituitary axis in vivo. ERalpha-/AA exhibited 69.9% lower serum LH levels compared with ERalpha-/- mice. Additionally, like wild type, ERalpha-/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERalpha-/AA mice. However, unlike wild type, both ERalpha-/AA and ERalpha-/- mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERalpha signaling is sufficient to convey a major portion of estrogen's negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERalpha signaling.

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.

Transcription factors, such as PDX-1, that normally mediate pancreatic development are capable of inducing hepatic progenitor cells to differentiate into cells with pancreatic islet characteristics. We hypothesized that simultaneous expression of multiple transcription factors involved in islet development might enhance the differentiation of hepatic progenitor cells. Bi- or tri-cistronic constructs were generated in hybrid adenovirus/adeno-associated virus (Ad/AAV) vectors containing neurogenin 3 (NGN3), BETA2 (NeuroD), and RIPE3b1 (MafA), each of which plays a role in islet cell differentiation. These vectors efficiently express multiple transcription factors and stimulate insulin promoter activity in a combinatorial manner. When these multi-cistronic constructs were administered in vivo, they induce hepatic expression of islet-specific markers, including PDX-1, insulin, glucagon, somatostatin, and islet-amyloid peptide. Administration of the Ad/AAV hybrid vectors to streptozotocin-induced diabetic mice reversed hyperglycemia, consistent the differentiation of functional hepatic insulin-secreting cells. These results indicate that Ad/AAV hybrid vectors can be used to administer combinations of factors that induce islet cell differentiation in hepatic progenitor cells.

Context: Steroidogenic factor 1 (SF1/AdBP4/FTZF1, NR5A1) is a nuclear receptor transcription factor that plays a key role in regulating adrenal and gonadal development, steroidogenesis, and reproduction. Targeted deletion of Nr5a1 (Sf1) in the mouse results in adrenal and gonadal agenesis, XY sex-reversal, and persistent Müllerian structures in males. Consistent with the murine phenotype, human mutations in SF1 were described initially in two 46,XY individuals with female external genitalia, Müllerian structures (uterus), and primary adrenal failure., Objective: Given recent case reports of haploinsufficiency of SF1 affecting testicular function in humans, we aimed to identify SF1 mutations in a cohort of individuals with a phenotypic spectrum of 46,XY gonadal dysgenesis/impaired androgenization (now termed 46,XY disorders of sex development) with normal adrenal function., Methods and Patients: The study included mutational analysis of NR5A1 in 30 individuals with 46,XY disorders of sex development, followed by functional studies of SF1 activity., Results: Heterozygous missense mutations in NR5A1 were found in four individuals (four of 30, 13%) with this phenotype. These mutations (V15M, M78I, G91S, L437Q) were shown to impair transcriptional activation through abnormal DNA binding (V15M, M78I, G91S), altered subnuclear localization (V15M, M78I), or disruption of the putative ligand-binding pocket (L437Q). Two mutations appeared to be de novo or germline changes. The other two mutations appeared to be inherited in a sex-limited dominant manner because the mother is heterozygous for the change., Conclusions: These studies demonstrate that SF1 mutations are more frequent than previously suspected causes of impaired fetal and postnatal testicular function in 46,XY individuals.

Context: Mutations in DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1; NR0B1) cause X-linked adrenal hypoplasia congenita, a disease characterized by primary adrenal failure, testicular dysgenesis, and gonadotropin deficiency. Most DAX1 mutations are deletions, nonsense, or frameshift mutations that markedly impair its transcriptional activity. Missense mutations have been restricted to the carboxy-terminal domain and are associated with more variable clinical phenotypes., Objective: The objective was to identify novel clinical phenotypes associated with DAX1 missense mutations., Patients and Design: We investigated the genetic basis of isolated mineralocorticoid deficiency in a patient who carries a unique missense mutation (W105C) in the amino-terminal region of DAX1., Results: The W105C DAX1 mutation in the proband was present in three asymptomatic hemizygous males, but it was not detected in the general population. Using in vitro studies of DAX1 expression and function in transfected cells, we demonstrate that the mutant DAX1 protein exhibits mild loss of function, whether studied for genes it represses or for genes it activates. Structure-function studies suggest that the W105C and other mutations in the aminoterminus are compensated by the presence of repeated LXXLL motifs that mediate DAX1 interactions with other proteins., Conclusions: We describe the first missense mutation in the aminoterminus of DAX1 and conclude that mutations in this region may be partially compensated by redundant functional domains. Mild DAX1 mutations may be a cause of isolated mineralocorticoid deficiency.

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERalpha, a mutant ERalpha (E207A/G208A) that selectively lacks ERE binding, or ERalpha null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17beta-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERalpha signaling. This proliferative response was confirmed by 5-bromo-2'-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathway-responsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERalpha (E207A/G208A) mutant. These results indicate that nonclassical ERalpha signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERalpha plays a critical physiologic role in the uterine response to estrogen.