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1.
F S Sci ; 4(2): 151-162, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37011812

RESUMEN

OBJECTIVE: To gain an understanding of the potential role of endoplasmic reticulum (ER) stress in the endometrial compartment during early pregnancy, a highly understudied area. DESIGN: This study examined the regulation of interferon-ß (IFNß) in response to ER stress in human decidualized and nondecidualized endometrial cells (human endometrial stromal cells [HESCs]) in vitro. In vivo, we examined ER stress and the IFNß levels locally in the mouse endometrium before and after implantation at embryonic day (E)1, E3, and E6. SETTING: The study was performed in a reproductive sciences laboratory for Human Growth and Development. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis allowed us to test the action of endogenous ER stress activation in the endometrial compartment likely triggered by implantation and its ability to increase the endometrial IFNß levels. RESULT(S): In vitro, we observed a significant difference in the IFNß levels in HESCs, in response to ER stress activation, where decidualized HESCs exhibited a threefold increase in the IFNß levels compared with nondecidualized HESCs. Apoptotic caspase-3 activation was also isolated to the decidualized cells as a result of ER stress-dependent suppression of nuclear factor-kappa beta-regulated antiapoptotic factors, XIAP and MCL-1. In vivo, mouse endometrial IFNß was present in F4/80-positive macrophages at all time points examined. After implantation (E6), the mouse luminal epithelial cells robustly coexpressed both IFNß and the ER stress marker immunoglobulin heavy chain binding protein (BiP). CONCLUSION(S): These analyses demonstrate that both in vivo and in vitro, differentiated and decidualized endometrial cells undergoing ER stress have the capacity to produce increased IFNß levels; therefore, ER stress activation in the endometrial compartment may play a vital role in promoting successful implantation events.


Asunto(s)
Implantación del Embrión , Endometrio , Embarazo , Femenino , Humanos , Animales , Ratones , Implantación del Embrión/fisiología , Diferenciación Celular , Interferón beta/metabolismo
2.
F S Sci ; 4(2): 141-150, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36603702

RESUMEN

OBJECTIVE: To examine the activation and consequence of uterine apoptotic caspase-3 action on 1 day after coitus (dpc) in the pregnant mouse. We have previously demonstrated that in a pregnant uterus, caspase-3 activation from mid to late gestation isolated to the myometrial compartment is largely nonapoptotic and controls uterine quiescence. Additionally, we had demonstrated that apoptotic caspase-3 activation isolated to the endometrial compartment at term regulated endometrial prostaglandin synthesis. DESIGN: Uteri were isolated from pseudopregnant and nonligated controls and unilateral and bilateral ligated uterine horn mouse models at 1, 3, and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining. Immunohistochemical analysis identified the site of uterine apoptotic caspase-3 activation. The truncated form of phospholipase A2 was examined as a measure of apoptotic caspase-3-mediated calcium independent phospholipase A2 (iPLA2) activation. RESULT(S): We identified the site and impact of uterine apoptotic caspase-3 activation using uteri isolated from nonpregnant control animals at estrous and diestrous and control pregnant mice at 1-19 dpc. Our analysis revealed that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral ligated uteri on 1 dpc and were not found in the pseudopregnant or bilateral ligated uterine horn or on 3 or 6 dpc in the control and unilateral ligated uteri. CONCLUSION(S): In this study, we determined that uterine caspase-3 activation on 1 dpc, which is endometrial and apoptotic in nature, may play a potential role in regulating the previously reported preimplantation surge in endometrial PGE2 synthesis through apoptotic caspase-3-mediated iPLA2 activation. Our data indicate that the presence of a conceptus on 1 dpc likely triggers an increase in endometrial apoptotic caspase-3-mediated iPLA2 activation. When activated, iPLA2 causes the hydrolysis of fatty acids, resulting in arachidonic acid release and PGE2 production, which has been demonstrated to act in a leutoprotective manner in early pregnancy, prolonging progesterone synthesis and promoting uterine receptivity.


Asunto(s)
Dinoprostona , Útero , Femenino , Embarazo , Ratones , Animales , Caspasa 3 , Endometrio , Fosfolipasas A2
3.
Dev Biol ; 477: 164-176, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34023333

RESUMEN

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.


Asunto(s)
Fertilidad/fisiología , Espermatozoides/fisiología , Factores de Transcripción/fisiología , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Femenino , Fertilidad/genética , Células HEK293 , Humanos , Masculino , Ratones Noqueados , Unión Proteica , ARN Mensajero/metabolismo , Espermatogénesis/genética , Espermatogénesis/fisiología , Factores de Transcripción/genética , Proteínas de Unión al GTP rab/fisiología
4.
Sci Rep ; 9(1): 4452, 2019 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-30872705

RESUMEN

The elevated level of Steroidogenic Factor 1 (Nr5a1, Sf-1) expression in the male gonadal development pathway, post sex determination, implies a vital role in testis gonadal differentiation. In this study we generated Sertoli cell-specific Nr5a1 KO mice (SC-SF-1-/-) at E14.5, which coincides with testis development post sex determination, using the Amh-Cre mouse model. Analysis of SC-SF-1-/- (Sertoli cell specific Nr5a1 knockout) testes demonstrated apoptosis as early as E15. Further analysis revealed that SC-SF-1-/- gonads displayed lower MDM2 levels resulting in elevated TP53 levels, which we believe may lead to apoptosis of the Sertoli cell population, inferring the possibility that NR5A1 directly regulates MDM2 expression. By E15.5, the Sertoli cell and germ cell population declined in SC-SF-1-/- mice resulting in the disruption of seminiferous cords with limited cord structure remaining at E18.5. Due to the loss of Sertoli and germ cells, the testis weights of SC-SF-1-/- mice at 6-weeks were much reduced; however, SC-SF-1-/- seminal vesicles weights were comparable suggesting intact Leydig cell androgen production. We conclude that NR5A1 regulates the TP53 pathway during development, is essential for fetal Sertoli cell survival and controls the cell cycle of Sertoli cells during differentiation.


Asunto(s)
Células de Sertoli/citología , Factor Esteroidogénico 1/metabolismo , Testículo/citología , Testículo/embriología , Animales , Hormona Antimülleriana/metabolismo , Apoptosis/genética , Supervivencia Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Integrasas/genética , Masculino , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factor de Transcripción SOX9/genética , Células de Sertoli/fisiología , Procesos de Determinación del Sexo , Factor Esteroidogénico 1/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
5.
Nucleic Acids Res ; 47(6): 2856-2870, 2019 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-30698747

RESUMEN

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.


Asunto(s)
Desarrollo Embrionario , Glucocorticoides/farmacología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Empalme del ARN/genética , Activación Transcripcional/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Embrión no Mamífero , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/genética , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Empalme del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transactivadores/fisiología , Activación Transcripcional/efectos de los fármacos , Pez Cebra
6.
EBioMedicine ; 39: 520-530, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30502052

RESUMEN

BACKGROUND: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. METHODS: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. FINDINGS: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. INTERPRETATION: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. FUND: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA).


Asunto(s)
Conexina 43/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Miometrio/metabolismo , Contracción Uterina/metabolismo , Empalme Alternativo , Línea Celular , Estrógenos/metabolismo , Exones , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Miometrio/citología , Especificidad de Órganos , Embarazo , Isoformas de Proteínas/metabolismo , Contracción Uterina/genética , Útero/metabolismo
7.
Cell Death Dis ; 9(10): 933, 2018 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-30224704

RESUMEN

The prevention of apoptotic caspase 3 activation through biological preconditioning, mediated through the modulation of the unfolded protein response has been demonstrated to ameliorate multiple pathophysiologies. The maintenance of non-apoptotic caspase 3 activity by the unfolded protein response within the pregnant uterus has previously been proven to be critical in inhibiting uterine myocyte contractility during pregnancy. Here we report that the pregnant uterus utilizes an unfolded protein response-preconditioning paradigm to conserve myometrial caspase 3 in a non-apoptotic state in order to effectively inhibit uterine contractility thereby preventing the onset of preterm labor. In the absence of appropriate endogenous preconditioning during pregnancy, uterine caspase 3 is transformed from a non-apoptotic to an apoptotic phenotype. Apoptotic caspase 3 activation results in the precocious triggering of local uterine inflammatory signaling and prostaglandin production, consequently resulting in an increased incidence of preterm birth. These findings represent a paradigm shift in our understanding of how preconditioning promotes the maintenance of uterine non-apoptotic caspase 3 action during pregnancy preventing the onset of premature uterine contraction and therefore defining the timing of the onset of labor.


Asunto(s)
Caspasa 3/metabolismo , Miometrio/citología , Miometrio/metabolismo , Respuesta de Proteína Desplegada/fisiología , Útero/citología , Útero/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Caspasa 3/genética , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Embarazo , Transducción de Señal/genética , Transducción de Señal/fisiología , Respuesta de Proteína Desplegada/genética
8.
J Cardiovasc Pharmacol Ther ; 22(4): 337-346, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28376665

RESUMEN

A broad definition of preconditioning is "the preparation for a subsequent action." Mounting evidence demonstrates that novel remote preconditioning paradigms, in which protective stimuli experienced locally can capacitate systemic tolerance and enhanced cell viability upon exposure to ensuing cellular insults, have been largely successful in the field of cardiovascular ischemia/reperfusion injury. To ensure successful protective preconditioning, some models (including the uterus) have been demonstrated to activate the unfolded protein response (UPR), which is a cellular stress response controlled at the level of the endoplasmic reticulum. However, in the context of remote preconditioning, activation of these intracellular molecular pathways must result in the extracellular transmission of adaptive signals to remote targets. In our recently published manuscript, we have described the activation of the UPR in the pregnant uterine myocyte to be associated with increased uterine myocyte quiescence and normal gestational length. We hypothesize that ubiquitous uterine gestational stresses experienced in every pregnancy, which have been demonstrated in other systems to activate the UPR, may induce a robust paracrine dissemination of a uterine secretome, for example, glucose-regulated protein 78, with preconditioning-like properties. Furthermore, we speculate that the gestational stress-induced uterine secretome acts to promote both local and systemic tolerance to the ensuing gestational insults, allowing for the maintenance of uterine quiescence. In this context, preterm labor may be the result of a pregnant uterus experiencing a stress it cannot accommodate or when it is unable to host an appropriate UPR resulting in insufficient preconditioning and a diminished local and systemic capacity to tolerate pregnancy-dependent increases in normal gestational stress. This is highly attractive from a clinical viewpoint as we ultimately aim to identify local and systemic adaptations that may serve as preconditioning stimuli for use as a strategy to restore appropriate preconditioning profiles to prolong uterine quiescence in pregnancy.


Asunto(s)
Precondicionamiento Isquémico/métodos , Nacimiento Prematuro/prevención & control , Contracción Uterina , Útero/fisiopatología , Adaptación Fisiológica , Animales , Velocidad del Flujo Sanguíneo , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Embarazo , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/fisiopatología , Flujo Sanguíneo Regional , Transducción de Señal , Respuesta de Proteína Desplegada , Útero/irrigación sanguínea , Útero/metabolismo
9.
Biol Reprod ; 95(6): 120, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27733380

RESUMEN

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.


Asunto(s)
Endometrio/metabolismo , Células Musculares/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína Desplegada/fisiología , Útero/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Femenino , Humanos , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Embarazo , Pliegue de Proteína , eIF-2 Quinasa/metabolismo
10.
J Vis Exp ; (111)2016 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-27286290

RESUMEN

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique - usually untranslated - first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Regiones Promotoras Genéticas , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Animales , Secuencia de Bases , Exones , Regulación de la Expresión Génica , Humanos , Ratones , ARN Mensajero/biosíntesis , ARN Mensajero/genética
11.
Proc Natl Acad Sci U S A ; 112(45): 14090-5, 2015 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-26504199

RESUMEN

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.


Asunto(s)
Caspasa 3/metabolismo , Caspasa 7/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Miometrio/fisiología , Embarazo/fisiología , Respuesta de Proteína Desplegada/fisiología , Útero/metabolismo , Análisis de Varianza , Animales , Línea Celular , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Ratones , Fenilbutiratos/farmacología , Embarazo/efectos de los fármacos , Progesterona/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
PLoS One ; 10(3): e0120783, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25803277

RESUMEN

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3' to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.


Asunto(s)
Fertilidad/genética , Receptores Androgénicos/genética , Espermatogénesis/genética , Transgenes/genética , Animales , Cromosomas Artificiales Bacterianos/genética , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Ratones , Receptores Androgénicos/deficiencia , Células de Sertoli/metabolismo , Testículo/citología , Testículo/metabolismo
13.
Biochim Biophys Acta ; 1829(12): 1288-99, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24189494

RESUMEN

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Unión al ADN/fisiología , Exones/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Células de Sertoli/metabolismo , Testículo/metabolismo , Factores de Transcripción/fisiología , Regiones no Traducidas 5' , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Especificidad de Órganos , Factores de Empalme de ARN , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/citología , Transcripción Genética/genética , Transfección
14.
Endocrinology ; 154(12): 4873-84, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24140717

RESUMEN

A successful postpartum involution permits the postnatal uterus to rapidly regain its prepregnancy function and size to ultimately facilitate an ensuing blastocyst implantation. This study investigates the molecular mechanisms that govern the initiation of the involution process by examining the signaling events that occur as the uterus transitions from the pregnant to postnatal state. Using mouse and baboon uteri, we found a remarkable cross-species conservation at the signal transduction level as the pregnant uterus initiates and progresses through the involution process. This study originated with the observation of elevated levels of caspase-3 activation in both the laboring mouse and baboon uterus, which we found to be apoptotic in nature as evidenced by the concurrent appearance of cleaved poly(ADP-ribose) polymerase. We previously defined a nonapoptotic and potential tocolytic role for uterine caspase-3 during pregnancy regulated by increased antiapoptotic signaling mediated by myeloid cell leukemia sequence 1 and X-linked inhibitor of apoptosis. In contrast, this study determined that diminished antiapoptotic signaling in the postpartum uterus allowed for both endometrial apoptotic and myometrial autophagic episodes, which we speculate are responsible for the rapid reduction in size of the postpartum uterus. Using our human telomerase immortalized myometrial cell line and the Simian virus-40 immortalized endometrial cell line (12Z), we demonstrated that the withdrawal of antiapoptotic signaling was also an upstream event for both the autophagic and apoptotic processes in the human uterine myocyte and endometrial epithelial cell.


Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Autofagia , Caspasa 3 , Línea Celular , Femenino , Etiquetado Corte-Fin in Situ , Trabajo de Parto/fisiología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Miometrio/citología , Papio anubis , Periodo Posparto , Embarazo , Transducción de Señal , Regulación hacia Arriba , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo
15.
PLoS One ; 8(9): e75152, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24058658

RESUMEN

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner.


Asunto(s)
Caspasa 3/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Células Musculares/enzimología , Miometrio/enzimología , Embarazo/fisiología , Transducción de Señal/fisiología , Animales , Activación Enzimática/fisiología , Femenino , Ratones , Células Musculares/citología , Miometrio/citología , Biosíntesis de Proteínas/fisiología , Transcripción Genética/fisiología
16.
Mol Endocrinol ; 26(2): 320-30, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22194343

RESUMEN

Our group has previously identified elevated levels of nonapoptotic active caspase 3 (CASP3) accompanied by increased prosurvival, antiapoptotic signaling in the pregnant mouse uterus during late gestation. We speculated that increased antiapoptotic signaling desensitized the pregnant uterine myocyte to the apoptotic action of uterine CASP3. This current study examines the mechanism by which the pregnant myocyte gains resistance to the apoptotic effects of increased uterine CASP3. Using both primary human pregnant fundal myometrial cultures and the telomerase-immortalized human uterine myocyte cell line (hTERT) as our model systems, uterine myocytes were exposed to UV irradiation and Fas ligand to stimulate both the intrinsic and extrinsic apoptotic pathways. Stimulation of either the intrinsic or extrinsic apoptotic pathways resulted in elevated levels of uterine myocyte CASP3. However, apoptotic cell death was restricted to CASP3 activated by intrinsic stimulation via UV light. In contrast Fas ligand-mediated CASP3 activation was accompanied by increased antiapoptotic signaling mimicking our in vivo observations in the pregnant mouse uterus. Using small interfering RNA to inhibit antiapoptotic signaling, we determined the ability of the human uterine myocyte to resist apoptotic cell death in the absence of the prosurvival, antiapoptotic signaling. Accordingly, suppression of antiapoptotic signaling specifically mediated by myeloid cell leukemia sequence 1 was sufficient to sensitize the uterine myocyte to undergo apoptotic cell death. These data demonstrate that elevated myeloid cell leukemia sequence 1 levels are sufficient to confer apoptotic resistance on the human uterine myocyte despite highly elevated levels of active CASP3.


Asunto(s)
Apoptosis , Caspasa 3/fisiología , Células Musculares/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Útero/citología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Catalasa/genética , Catalasa/metabolismo , Línea Celular , Fragmentación del ADN , Activación Enzimática , Proteína Ligando Fas/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Células Musculares/enzimología , Células Musculares/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Embarazo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-bcl-2/genética , Rayos Ultravioleta
17.
Biol Reprod ; 85(2): 417-24, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21566000

RESUMEN

Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pregnant uterine myocyte may harness the tocolytic activity of active caspases while avoiding apoptotic cell death. Using the pregnant mouse model, we have analyzed the uterus for changes in pro- and antiapoptotic signaling patterns associated with the advancing stages of pregnancy. Briefly, we have found that members of the IAP family, such as SURVIVIN and XIAP, and the Bcl2 family members, such as MCL1, are elevated in the uterine myocyte during late gestation. The IAP family members are the only endogenous inhibitors of active caspase 3, and MCL1 limits activation of caspase 3 by suppressing proapoptotic signaling. Elevated XIAP levels partner with SURVIVIN, resulting in increased levels of the antiapoptotic MCL1 via NFKB activation; these together have the potential to limit both the activity and level of active caspase 3 in the pregnant uterus as term approaches. We propose that modification of these antiapoptotic signaling partners allows the pregnant uterus to escape the apoptotic action of elevated active caspase 3 levels but also functions to limit the levels of active uterine caspase 3 near term.


Asunto(s)
Apoptosis/fisiología , Caspasa 3/metabolismo , FN-kappa B/metabolismo , Útero/fisiología , Animales , Caspasa 3/genética , ADN/metabolismo , Femenino , Genoma , Ratones , FN-kappa B/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Embarazo , Útero/citología
18.
Endocrinology ; 152(1): 93-102, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21106871

RESUMEN

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11ß-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11ß-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11ß-HSD1/H6PDH double-KO (DKO) mice, in which 11ß-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11ß-HSD1KO mice. Critically, in contrast to 11ß-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11ß-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Deshidrogenasas de Carbohidratos/metabolismo , Homeostasis/fisiología , Músculo Esquelético/fisiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Glucemia , Deshidrogenasas de Carbohidratos/genética , Corticosterona/sangre , Regulación Enzimológica de la Expresión Génica , Insulina/sangre , Ratones , Ratones Noqueados , Enfermedades Musculares/enzimología , Enfermedades Musculares/genética
19.
Biol Reprod ; 80(5): 928-34, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19144964

RESUMEN

The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. Using the mouse as our model system, we examined, by Western blot analysis, levels of active caspase 3 and its association with the degradation of uterine contractile proteins during pregnancy. Our data demonstrate that caspase 3-specific cleavage fragments of uterine myocyte contractile proteins are elevated in late gestation. Prior to the onset of labor, active caspase 3 levels and fragmentation of the uterine myocyte contractile proteins decline. We postulate that uterine caspase 3 acts as an anticontractile agent maintaining uterine quiescence through degradation of uterine contractile proteins during late pregnancy. We propose that decreased progesterone action during the final days of pregnancy controls the timing of the onset of uterine contractions by removing the anticontractile action of the apoptotic protein caspase 3 locally in the pregnant myometrium.


Asunto(s)
Caspasa 3/metabolismo , Progesterona/metabolismo , Útero/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Secuencia de Bases , Caspasa 3/genética , Femenino , Humanos , Inicio del Trabajo de Parto/metabolismo , Ratones , Ratones Endogámicos ICR , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Embarazo , Progesterona/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Contracción Uterina/metabolismo , Útero/efectos de los fármacos
20.
Mol Endocrinol ; 22(4): 951-64, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18187601

RESUMEN

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR-/-), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR-/- mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR-/- mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.


Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Mitocondrias/metabolismo , Fosfoproteínas/fisiología , Glándulas Suprarrenales/metabolismo , Hormona Adrenocorticotrópica/sangre , Animales , Southern Blotting , Corticosterona/sangre , Femenino , Técnicas de Transferencia de Gen , Gónadas/metabolismo , Immunoblotting , Masculino , Ratones , Ratones Transgénicos , Modelos Genéticos , Ovario/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismo
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