RESUMEN
The Notch pathway is a contact-dependent, or juxtacrine, signaling system that is conserved in metazoan organisms and is important in many developmental processes. Recent investigations have demonstrated that the Notch pathway is active in both the embryonic and postnatal ovary and plays important roles in events including follicle assembly and growth, meiotic maturation, ovarian vasculogenesis and steroid hormone production. In mice, disruption of the Notch pathway results in ovarian pathologies affecting meiotic spindle assembly, follicle histogenesis, granulosa cell proliferation and survival, corpora luteal function and ovarian neovascularization. These aberrations result in abnormal folliculogenesis and reduced fertility. The knowledge of the cellular interactions facilitated by the Notch pathway is an important area for continuing research, and future studies are expected to enhance our understanding of ovarian function and provide critical insights for improving reproductive health. This review focuses on the expression of Notch pathway components in the ovary, and on the multiple functions of Notch signaling in follicle assembly, maturation and development. We focus on the mouse, where genetic investigations are possible, and relate this information to the human ovary.
Asunto(s)
Mamíferos/metabolismo , Ovario/metabolismo , Ovario/patología , Receptores Notch/metabolismo , Animales , Femenino , Humanos , Transducción de SeñalRESUMEN
Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Cristalografía por Rayos X/métodos , AMP Cíclico/química , Cisteína/química , ADN/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Leucina Zippers , Datos de Secuencia Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Activación TranscripcionalRESUMEN
The murine primordial follicle pool develops largely within 3 days after birth through germline nest breakdown and enclosure of oocytes within pregranulosa cells. The mechanisms that trigger primordial follicle formation likely are influenced by a transition from the maternal to fetal hormonal milieu at the time of birth. High levels of maternal estrogen maintain intact germline nest in fetal ovary, and decrease of estrogen after birth is permissive of follicle formation. In the present study, we measured an increase in neonatal serum follicle-stimulating hormone (FSH), which corresponded to falling estradiol (E(2)) levels during the critical window of primordial follicle formation (Postnatal Days 1-3). To determine whether fetal hormones contribute in an active manner to primordial follicle formation, mouse fetal ovaries (17.5 days postcoitus) were cultured in vitro at two concentrations of E(2) (meant to reflect maternal and fetal levels of E(2)) and FSH for 6 days. High levels of E(2) (10(-6) M) inhibited germline nest breakdown, and this effect was significantly reduced when fetal ovaries were cultured in the low E(2) concentration (10(-10) M). FSH facilitated germline nest breakdown and primordial follicle formation under both high and low E(2) culture conditions. Low E(2) was identified as being more permissive for the effects of FSH on primordial follicle formation by stimulating the up-regulation of Fshr and activin beta A subunit (Inhba) expression, pregranulosa cell proliferation, and oocyte survival. The decrease of E(2) plus the presence of FSH after birth are critical for primordial follicle formation and the expression of oocyte-specific transcription factors (Figla and Nobox) in that inappropriate exposure to FSH or E(2) during follicle formation resulted in premature or delayed primordial folliculogenesis. In conclusion, with the drop of E(2) level after birth, FSH promotes primordial follicle formation in mice by stimulating local activin signaling pathways and the expression of oocyte-specific transcription factors.
Asunto(s)
Estradiol/fisiología , Hormona Folículo Estimulante/fisiología , Folículo Ovárico/fisiología , Activinas/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Proliferación Celular , Supervivencia Celular , AMP Cíclico/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Oocitos/fisiología , Técnicas de Cultivo de Órganos , EmbarazoRESUMEN
The Notch pathway plays diverse and complex roles in cell signaling during development. In the mammalian ovary, Notch is important for the initial formation and growth of follicles, and for regulating the proliferation and differentiation of follicular granulosa cells during the periovulatory period. This study seeks to determine the contribution of female germ cells toward the initial activation and subsequent maintenance of Notch signaling within somatic granulosa cells of the ovary. To address this issue, transgenic Notch reporter (TNR) mice were crossed with Sohlh1-mCherry (S1CF) transgenic mice to visualize Notch-active cells (EGFP) and germ cells (mCherry) simultaneously in the neonatal ovary. To test the involvement of oocytes in activation of Notch signaling in ovarian somatic cells, we ablated germ cells using busulfan, a chemotherapeutic alkylating agent, or investigated KitWv/Wv (viable dominant white-spotting) mice that lack most germ cells. The data reveal that Notch pathway activation in granulosa cells is significantly suppressed when germ cells are reduced. We further demonstrate that disruption of the gene for the Notch ligand Jag1 in oocytes similarly impacts Notch activation and that recombinant JAG1 enhances Notch target gene expression in granulosa cells. These data are consistent with the hypothesis that germ cells provide a ligand, such as Jag1, that is necessary for activation of Notch signaling in the developing ovary.
Asunto(s)
Células de la Granulosa/metabolismo , Proteína Jagged-1/metabolismo , Oocitos/metabolismo , Receptores Notch/metabolismo , Animales , Línea Celular , Femenino , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: The TGF-beta family protein activin has numerous reported activities with some uncertainty in the reproductive axis and development. The precise roles of activin in in vivo system were investigated using a transient gain of function model. METHODS: To this end, an expression plasmid, pCMV-rAct, with the activin betaA cDNA fused to the cytomegalovirus promoter, was introduced into muscle of the female adult mice by direct injection. RESULTS: Activin betaA mRNA was detected in the muscle by RT-PCR and subsequent Southern blot analysis. Activin betaA was also detected, and western blot analysis revealed a relatively high level of serum activin with correspondingly increased FSH. In the pCMV-rAct-injected female mice, estrus stage within the estrous cycle was extended. Moreover, increased numbers of corpora lutea and a thickened granulosa cell layer with a small antrum in tertiary follicles within the ovary were observed. When injected female mice were mated with males of proven fertility, a subset of embryos died in utero, and most of those that survived exhibited increased body weight. CONCLUSION: Taken together, our data reveal that activin betaA can directly influence the estrous cycle, an integral part of the reproduction in female mice and activin betaA can also influence the embryo development as an endocrine fashion.
Asunto(s)
Desarrollo Embrionario , Ciclo Estral/fisiología , Subunidades beta de Inhibinas/genética , Ovario/crecimiento & desarrollo , Activinas/sangre , Animales , Southern Blotting , Peso Corporal , Femenino , Hormona Folículo Estimulante/sangre , Inyecciones Intramusculares , Masculino , Ratones , Ratones Endogámicos ICR , Ovario/citología , Plásmidos/administración & dosificación , Embarazo , ARN Mensajero/metabolismo , Reproducción/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/citología , Factores de TiempoRESUMEN
The GHRH receptor is expressed in the somatotroph cell of the anterior pituitary, where it functions to mediate GHRH-stimulated GH release. To study pituitary and somatotroph cell-specific expression of this gene, a transgenic mouse model and complementary cell culture experiments were developed. The activity of the 1.6-kb proximal rat GHRH receptor promoter was examined in vivo by generating transgenic mice with the promoter directing expression of a luciferase reporter. The promoter directs tissue-specific expression; luciferase is highly expressed in the pituitary but absent from 14 other tissues. Immunocytochemistry experiments show that transgene expression is targeted to GH-expressing somatotroph cells. The transgene is 5-fold more highly expressed in males than females, and there is an increase in transgene expression leading up to the onset of puberty. The 1.6-kb promoter was further examined in cell culture experiments, which revealed that the promoter is selectively activated in pituitary cells and that promoter-reporter expression in nonpituitary cells can be enhanced by the pituitary-specific transcription factor Pit-1. EMSAs identified 10 short regions that specifically bind Pit-1 with highly variable relative affinities. The highest affinity site was previously identified and is required for Pit-1 activation of the promoter. Four additional sites contribute to Pit-1 regulation of the promoter and are important to achieving full activation of the gene. The results show that the 1.6-kb promoter is sufficient to direct tissue- and cell-specific expression in vivo and is regulated by Pit-1.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Adenohipófisis/metabolismo , Receptores de Neuropéptido/biosíntesis , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/biosíntesis , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Factor de Transcripción Pit-1/fisiología , Animales , Animales Modificados Genéticamente , Genes Reporteros , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Ratas , TransgenesRESUMEN
The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteína Jagged-1/metabolismo , Sistema de Señalización de MAP Quinasas , Receptor Notch2/agonistas , Receptor Notch3/agonistas , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/farmacología , Estradiol/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Gonadotropinas Equinas/farmacología , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/antagonistas & inhibidores , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína Jagged-1/antagonistas & inhibidores , Proteína Jagged-1/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos , Ratones Transgénicos , Progesterona/metabolismo , Interferencia de ARN , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.
Asunto(s)
Receptores de Activinas/genética , Activinas/genética , Estradiol/farmacología , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/fisiología , Transducción de Señal/efectos de los fármacos , Receptores de Activinas/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Activinas/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Dietilestilbestrol/farmacología , Estrógenos no Esteroides/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Inmunohistoquímica , Inhibinas/genética , Ratones , Ratones Endogámicos , Tamaño de los Órganos , Folículo Ovárico/citología , Embarazo , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Chronic ovulation as a contributing factor for the development of epithelial ovarian cancer in women has long been an outstanding hypothesis. To test the incessant ovulation hypothesis, mice were superovulated using weekly ip injections of pregnant mare serum gonadotropin (5 IU/animal), followed 48 h later by human chorionic gonadotropin (5 IU/animal). Wild-type CD1 mice were used along with CD1 mice expressing a Smad2 dominant-negative (Smad2DN) transgene under the control of the Müllerian inhibiting substance promoter that targets expression to the ovary and enhances cyst formation. After chronic injections, ovaries were analyzed from animals 6 months of age for the total adjusted number of cysts, cyst area, cyst location, and key signaling pathways. All observed cysts were confirmed to be of epithelial origin. The number of cysts was not significantly different between superovulated and control mice in either the wild-type or Smad2DN groups. However, the combination of the Smad2DN transgene and superovulation resulted in an increase in cyst formation compared with normal littermates that were unstimulated. Rapid proliferation of the cells lining the cysts was detected using bromodeoxyuridine and phospho-histone 3 immunohistochemistry but was not different in the ovarian surface epithelium or in the cyst lining between groups. These data suggest that chronic superovulation in Smad2DN mice results in a higher incidence of cyst formation compared with unstimulated controls, but the epithelial lined cysts did not progress to cancer over the course of this study.
Asunto(s)
Quistes Ováricos/fisiopatología , Ovulación/fisiología , Proteína Smad2/genética , Proteína Smad2/metabolismo , Superovulación/fisiología , Activinas/metabolismo , Animales , División Celular/fisiología , Enfermedad Crónica , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Gonadotropinas Equinas/farmacología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Quistes Ováricos/complicaciones , Quistes Ováricos/patología , Neoplasias Ováricas/etiología , Neoplasias Ováricas/patología , Neoplasias Ováricas/fisiopatología , Fosforilación , Transducción de Señal/fisiología , Superovulación/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This 'cutting edge' editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.
Asunto(s)
Reproducción/fisiología , Sistema Urogenital/anatomía & histología , Sistema Urogenital/fisiología , Sistema Endocrino , Femenino , Humanos , Folículo Ovárico/anatomía & histología , Folículo Ovárico/crecimiento & desarrollo , Transducción de SeñalRESUMEN
The inhibin alpha-subunit gene is transcriptionally activated by FSH in ovarian granulosa cells during follicular growth. We have investigated the roles of the NR5A family nuclear receptors steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) in transcriptional activation of the inhibin alpha-subunit gene. Transfection assays using an inhibin alpha-subunit promoter reporter in GRMO2 granulosa cells show that LRH-1 and SF-1 act similarly to increase promoter activity, and that the activity of both transcription factors is augmented by the coactivators cAMP response element-binding protein-binding protein and steroid receptor coactivator 1. However, chromatin immunoprecipitation experiments illustrate differential dynamic association of LRH-1 and SF-1 with the alpha-subunit inhibin promoter in both primary cells and the GRMO2 granulosa cell line such that hormonal stimulation of transcription results in an apparent replacement of SF-1 with LRH-1. Transcriptional stimulation of the inhibin alpha-subunit gene is dependent on MAPK kinase activity, as is the dynamic association/disassociation of SF-1 and LRH-1 with the promoter. Inhibition of the phosphatidylinositol 3-kinase signaling pathway influences promoter occupancy and transcriptional activation by SF-1 but not LRH-1, suggesting a possible mechanistic basis for the distinct functions of these NR5A proteins in inhibin alpha-subunit gene regulation.
Asunto(s)
Células de la Granulosa/metabolismo , Proteínas de Homeodominio/metabolismo , Inhibinas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Histona Acetiltransferasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Coactivador 1 de Receptor Nuclear , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Regiones Promotoras Genéticas , Factor Esteroidogénico 1RESUMEN
The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.
Asunto(s)
Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Inhibinas/genética , Animales , Sitios de Unión , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , Dimerización , Regulación hacia Abajo , Femenino , Gonadotropinas/farmacología , Células de la Granulosa/metabolismo , Humanos , Inhibinas/efectos de los fármacos , Inhibinas/metabolismo , Hormona Luteinizante/metabolismo , Hormona Luteinizante/farmacología , Mutación , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas , Ratas , Ratas Sprague-Dawley , Elementos de RespuestaRESUMEN
Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-alpha gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.
Asunto(s)
ADN/genética , ADN/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Homeodominio/genética , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Sustancias Macromoleculares , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Factor Esteroidogénico 1 , Factores de Transcripción/genéticaRESUMEN
GHRH is a hypothalamic peptide that stimulates the synthesis and secretion of GH from pituitary somatotroph cells. The GHRH receptor is a seven-transmembrane G protein-coupled receptor that localizes to the surface of somatotroph cells and binds GHRH. Alternative splicing of the GHRH receptor primary transcript at the intron/exon boundary 3' of exon 11 results in inclusion of sequence that is normally intronic. In the human, this inclusion has an in-frame premature stop codon, and this variant mRNA encodes a protein truncated just before the sixth transmembrane domain. To identify the effects of the truncated receptor on signaling of the wild-type receptor and the mechanisms by which its effects are produced, the full-length and truncated receptor constructs were epitope tagged and transfected into HeLa T4 cells to examine signaling and expression. Results show that the truncated GHRH receptor cannot signal through the cAMP pathway and acts as a dominant inhibitor of wild-type receptor signaling. The wild-type and truncated GHRH receptor proteins form a complex. Stably transfected cell lines were generated to examine the mechanism of signal inhibition by the truncated receptor. The data show that receptor cell surface expression is not altered when the wild-type and truncated receptors are cotransfected, but that truncated receptor coexpression substantially reduces GHRH binding by the wild-type receptor. The results support an important role for alternative splicing in mediating the effects of G protein-coupled receptors in general, and suggest that the GHRH receptor can form multimers, which may be important to its signaling properties.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/metabolismo , Receptores de Neuropéptido/fisiología , Receptores de Hormona Reguladora de Hormona Hipofisaria/fisiología , Empalme Alternativo , AMP Cíclico/biosíntesis , Dimerización , Hormona del Crecimiento/metabolismo , Células HeLa , Humanos , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Transducción de Señal , TransfecciónRESUMEN
The ovarian surface epithelium (OSE) is a monolayer of cells that surround the ovary and accommodate repeated tear and repair in response to ovulation. OSE cells are thought to be the progenitors of 90% of ovarian cancers. Currently, the total amount of proliferation of the OSE has not been reported in response to one ovulatory event. In this study, proliferation of the OSE was quantified in response to superovulation induced by ip injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) in immature 27-d-old CD1 mice using bromodeoxyuridine (BrdU). BrdU incorporation into the OSE cells was measured from the time of hCG injection for a total cumulative label of 12 h. BrdU incorporation was also measured from the time of PMSG injection for a total label of 60 h to correlate proliferation with specific gonadotropin stimulation. The OSE proliferation was significantly higher in superovulated animals compared with control mice at all time points. Proliferation was also analyzed in discrete anatomical sections and indicated that OSE covering antral follicles and corpora lutea proliferated more rapidly than OSE distal to follicular growth. Finally, apoptosis was assessed in response to ovulation, and virtually no cell death within the OSE was detected. These data demonstrate that the OSE, especially near antral follicles and corpora lutea, proliferates significantly in response to the gonadotropins PMSG and hCG. Therefore, ovarian surface cell division in response to ovulation could contribute to ovarian cancer by proliferation-induced DNA mutations and transformed cell progression.
Asunto(s)
Células Epiteliales/citología , Gonadotropinas/metabolismo , Ovario/metabolismo , Animales , Apoptosis , Bromodesoxiuridina/farmacología , Proliferación Celular , Cuerpo Lúteo/metabolismo , Femenino , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Modelos Biológicos , Modelos Estadísticos , Mutación , Folículo Ovárico/metabolismo , Ovulación , Superovulación , Factores de TiempoRESUMEN
Inhibin is a dimeric peptide hormone produced in ovarian granulosa cells that suppresses FSH synthesis and secretion in the pituitary. Expression of inhibin alpha- and beta-subunit genes in the rodent ovary is positively regulated by FSH and negatively regulated after the preovulatory LH surge. We have investigated the role of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta) in repressing the inhibin alpha-subunit gene. C/EBPbeta knockout mice fail to appropriately down-regulate inhibin alpha-subunit mRNA levels after treatment with human chorionic gonadotropin, indicating that C/EBPbeta may function to repress inhibin gene expression. The expression and regulation of C/EBPbeta were examined in rodent ovary, and these studies show that C/EBPbeta is expressed in ovary and granulosa cells and is induced in response to human chorionic gonadotropin. Transient cotransfections with an inhibin promoter-luciferase reporter in a mouse granulosa cell line, GRMO2 cells, show that C/EBPbeta is capable of repressing both basal and forskolin-stimulated inhibin gene promoter activities. An upstream binding site for C/EBPbeta in the inhibin alpha-subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/EBPbeta also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element (CRE). Electrophoretic mobility shift assays show that C/EBPbeta effectively competes with CRE-binding protein for binding to this atypical CRE. Thus, there are two distinct mechanisms by which C/EBPbeta represses inhibin alpha-subunit gene expression in ovarian granulosa cells.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Regulación de la Expresión Génica , Inhibinas/genética , Animales , Sitios de Unión , Modulador del Elemento de Respuesta al AMP Cíclico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/fisiología , Femenino , Ratones , Ovario/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Pelvic pain is a common presenting ailment in women often linked to ovulation, endometriosis, early pregnancy, ovarian cancer, and cysts. Clear differential diagnosis for each condition caused by these varied etiologies is difficult and may slow the delivery of therapy that, in the case of ovarian cancer, could be fatal. Ovarian endosalpingiosis, a pelvic condition typified by the presence of cystic glandular structures lined by benign tubal/salpingeal epithelium, is also associated with pelvic pain in women. The exact cellular antecedents of these epithelial lined cystic structures are not known, nor is there a known link to ovarian cancer. A mouse model of ovarian endosalpingiosis has been developed by directing a dominant-negative version of the TGF-beta transcription factor, Smad2, to the ovary using the Müllerian-inhibiting substance promoter (MIS-Smad2-dn). Female mice develop an ovarian endosalpingeal phenotype as early as 3 months of age. Importantly, cysts continuous with the ovarian surface epithelial have been identified, indicating that these cyst cells may be derived from the highly plastic ovarian surface epithelial cell layer. A second transgenic mouse model that causes loss of activin action (inhibin alpha-subunit transgenic mice) develops similar cystic structures, supporting a TGF-beta/activin/Smad2 dependence in the onset of this disease.
Asunto(s)
Coristoma/patología , Quistes/patología , Modelos Animales de Enfermedad , Trompas Uterinas , Enfermedades del Ovario/patología , Activinas/antagonistas & inhibidores , Animales , Hormona Antimülleriana , Femenino , Fertilidad , Hormona Folículo Estimulante/sangre , Expresión Génica , Marcación de Gen/métodos , Genes Dominantes , Glicoproteínas/genética , Inhibinas/genética , Ratones , Ratones Transgénicos , Enfermedades del Ovario/genética , Ovario/patología , Fenotipo , Regiones Promotoras Genéticas , Proteína Smad2/genética , Hormonas Testiculares/genética , TransgenesRESUMEN
Originally, activins were identified as stimulators of FSH release in reproduction. Other activities, including secondary axis formation in development, have since been revealed. Here, we investigated the influence of activin ßA on the body, including the gastro-intestinal (GI) tract. Initially, the activin ßA protein was detected in the serum proportional to the amount of pCMV-rAct plasmid injected. The induced level of activin ßA in muscle was higher in female than male mice. Subsequent results revealed that stomach and intestine were severely damaged in pCMV-rAct-injected mice. At the cellular level, loss of parietal cells was observed, resulting in increased pH within the stomach. This phenomenon was more severe in male than female mice. Consistent with damage of the stomach and intestine, activin ßA often led to necrosis in the tip of the tail or foot, and loss of body weight was observed in pCMV-rAct-injected male but not female mice. Finally, in pCMV-rAct-injected mice, circulating activin ßA led to death at supraphysiological doses, and this was dependent on the strain of mice used. Taken together, these results indicate that activin ßA has an important role outside of reproduction and development, specifically in digestion. These data also indicate that activin ßA must be controlled within a narrow range because of latent lethal activity. In addition, our approach can be used effectively for functional analysis of secreted proteins.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/patología , Subunidades beta de Inhibinas/genética , Plásmidos/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Femenino , Concentración de Iones de Hidrógeno , Subunidades beta de Inhibinas/sangre , Masculino , Ratones , Músculos/metabolismo , Necrosis , Plásmidos/genética , Factores Sexuales , Pérdida de PesoRESUMEN
GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.
Asunto(s)
AMP Cíclico/biosíntesis , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/metabolismo , Hormonas Peptídicas/farmacología , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Animales , Benzazepinas/farmacología , Colforsina/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Ghrelina , Células HeLa , Hemaglutininas/genética , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Técnicas de Inmunoadsorción , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Receptores de la Hormona Gastrointestinal/efectos de los fármacos , Receptores de la Hormona Gastrointestinal/fisiología , Receptores de Ghrelina , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Receptores de Péptido Intestinal Vasoactivo/fisiología , Proteínas Recombinantes de Fusión , Secretina/farmacología , Transducción de Señal , Tetrazoles/farmacología , Transfección , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Inhibin is a gonadal hormone composed of an a-subunit and one of two beta-subunits (betaA, betaB), and its primary role is to inhibit FSH secretion by the pituitary. To investigate the roles of inhibin alpha in the reproductive system, an expression plasmid, pCMV-rINA, with the rat inhibin alpha cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Inhibin alpha mRNA was detected in the muscle by RT-PCR and Southern blot analysis. Inhibin protein was also detected, and Western blot analysis revealed a relatively high level of serum inhibin, but not of activin betaA. The estrous cycle of the pCMV-rINA-injected mice was extended, but there was no change in levels of pituitary FSH mRNA or serum FSH and no ovarian cysts were observed. When injected female mice were mated with males of proven fertility, litter size increased. Surprisingly, the embryos of pregnant females injected with pCMV-rINA, were retarded in growth and had defects in internal organs. When male mice were injected, testicle weight increased slightly without any noticeable change in the histology of the seminiferous tubules. Taken together, our data indicate that the inhibin alpha subunit influences a number of the reproductive functions of female mice.