RESUMO
Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.
Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Trabalho de Parto Prematuro/genética , Ovário/metabolismo , Progesterona/biossíntese , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia , Proteína Beclina-1 , Vias Biossintéticas/genética , Endossomos/metabolismo , Endossomos/ultraestrutura , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Células Lúteas/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , GravidezRESUMO
Autophagy is a major catabolic process in which intracellular membrane structures, protein complexes, and lysosomes are formed as lysoautophagosome to degrade and renew cytoplasmic components. Autophagy is physiologically a strategy and mechanism for cellular homeostasis as well as adaptation to stress, and thus alterations in the autophagy machinery may lead to diverse pathological conditions. The role of autophagy in cancer is complex, and the current literature reflects this as a 'double-edged sword'. Autophagy shows promise as a novel therapeutic target in various types of breast cancer, inhibiting or increasing treatment efficacy in a context- and cell-type-dependent manner. This review aims to summarize the recent advances in the understanding of the mechanisms by which key modulators of autophagy participate in cancer metastasis, highlight different autophagy-deficient murine models for breast cancer study, and provide further impetus for the modulation of autophagy in anticancer therapy.
Assuntos
Autofagia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Antineoplásicos/química , Proteína Beclina-1/metabolismo , Sobrevivência Celular , Genótipo , Humanos , Inflamação , Lisossomos/metabolismo , Camundongos , Metástase Neoplásica , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Bcl-x is the most abundantly expressed member of the Bcl-2 gene family in macrophages, but its role in macrophage apoptosis during atherogenesis is unknown. METHODS AND RESULTS: We previously reported dual pro- and antiatherogenic effects of macrophage survival in early versus advanced atherosclerotic lesions, respectively, potentially reflecting growing impairment of efferocytosis during plaque progression. Here, we specifically inactivated Bcl-x in macrophages and evaluated its impact on atherosclerotic lesion formation in Apoe(-/-) mice at various stages of the disease. Bcl-x deficiency in macrophages increased their susceptibility to apoptosis, resulting in the depletion of tissue macrophages in vivo, including its major pool, Küppfer cells in the liver. We also observed increased cholesterol levels that were, however, not associated with any acceleration of early atherosclerotic plaque progression. This observation suggests that the atheroprotective effect of macrophage apoptosis at that stage of disease was counterbalanced by enhanced cholesterol levels. Bcl-x KO(mac)/Apoe(-/-) mice exhibited significantly larger advanced lesions than control mice. These lesions showed vulnerable traits. Such enhanced lesion size may occur as a result not only of apoptotic cell accumulation but also of elevated cholesterol levels. CONCLUSIONS: Modulation of macrophage resistance to apoptosis through targeted deletion of Bcl-x has a major impact on the entire macrophage cell population in the body, including Küpffer cells. Macrophage survival may, therefore, not only influence atherosclerotic plaque development and vulnerability but also cholesterol metabolism.
Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/genética , DNA/genética , Regulação da Expressão Gênica , Proteína bcl-X/genética , Animais , Apolipoproteínas E/genética , Apoptose , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Progressão da Doença , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Proteína bcl-X/biossínteseRESUMO
The human endometrium shows a remarkable regenerative capacity that enables cyclical regeneration and remodeling throughout a woman's reproductive life. Although early postnatal uterine developmental cues direct this regeneration, the vital factors that govern early endometrial programming are largely unknown. We report that Beclin-1, an essential autophagy-associated protein, plays an integral role in uterine morphogenesis during the early postnatal period. We show that conditional depletion of Beclin-1 in the uterus triggers apoptosis and causes progressive loss of Lgr5+/Aldh1a1+ endometrial progenitor stem cells, with concomitant loss of Wnt signaling, which is crucial for stem cell renewal and epithelial gland development. Beclin-1 knockin (Becn1 KI) mice with disabled apoptosis exhibit normal uterine development. Importantly, the restoration of Beclin-1-driven autophagy, but not apoptosis, promotes normal uterine adenogenesis and morphogenesis. Together, the data suggest that Beclin-1-mediated autophagy acts as a molecular switch that governs the early uterine morphogenetic program by maintaining the endometrial progenitor stem cells.
Assuntos
Endométrio , Útero , Animais , Feminino , Humanos , Camundongos , Gravidez , Autofagia , Proteína Beclina-1 , Células-TroncoRESUMO
The mammary gland is a developmentally dynamic, hormone-responsive organ that undergoes proliferation and differentiation within the secretory epithelial compartment during pregnancy. The epithelia are maintained by pro-survival signals (e.g., Stat5, Akt1) during lactation, but undergo apoptosis during involution through inactivation of cell survival pathways and upregulation of pro-apoptotic proteins. To assess if the survival signals in the functionally differentiated mammary epithelial cells can override a pro-apoptotic signal, we generated transgenic mice that express Bax under the whey acidic protein (WAP) promoter. WAP-Bax females exhibited a lactation defect and were unable to nourish their offspring. Mammary glands demonstrated: (1) a reduction in epithelial content, (2) hallmark signs of mitochondria-mediated cell death, (3) an increase in apoptotic cells by TUNEL assay, and (4) precocious Stat3 activation. This suggests that upregulation of a single pro-apoptotic factor of the Bcl-2 family is sufficient to initiate apoptosis of functionally differentiated mammary epithelial cells in vivo.
Assuntos
Apoptose , Glândulas Mamárias Animais/embriologia , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Epiteliais/fisiologia , Feminino , Vetores Genéticos , Imuno-Histoquímica , Lactação/genética , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT5/metabolismo , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
The WNTs are secreted proteins that control essential developmental processes, such as embryonic patterning, cell growth, migration, and differentiation. In mice, three members of the Wnt gene family (Wnt4, Wnt5a, and Wnt7a) have been studied extensively in the female reproductive tract. The present study determined effects of postnatal day and exposure to diethylstilbestrol (DES) on Wnt and Fzd gene expression in the mouse uterus as well as the biological role of Wnt11 in postnatal mouse uterine development and function. Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd6, and Fzd10 were detected by in situ hybridization in the neonatal mouse uterus. In situ hybridization analyses revealed that Wnt4, Wnt5a, and Wnt16 were localized in the endometrial stroma, whereas Wnt7a, Wnt7b, Wnt11, Fzd6, and Fzd10 were in the uterine epithelia of neonatal mice. Exposure of mice to estrogen or estrogen receptor agonists during critical development periods inhibits endometrial adenogenesis. In the present study, DES-induced disruption of endometrial gland development was associated with reduction or suppression of Wnt4, Wnt5a, Wnt7a, Wnt11, Wnt16, and Fzd10. Ablation of Wnt11, an epithelial-expressed, DES-regulated gene, in the neonatal uterus did not affect endometrial adenogenesis or expression of other Wnt genes. Interestingly, Wnt11-deleted uteri had more endometrial glands on Postnatal Day 10. Although CTNNB1 expression was not affected by ablation of Wnt11, Vangl2 was inhibited in the uteri of Wnt11(d/d) mice. These results support the idea that a number of different Wnt genes are potential regulators for uterine morphogenesis; however, Wnt11 does not have a direct effect on uterine development.
Assuntos
Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Endométrio/crescimento & desenvolvimento , Receptores Frizzled/metabolismo , Morfogênese , Proteínas Wnt/metabolismo , Envelhecimento/metabolismo , Animais , Dietilestilbestrol/farmacologia , Endométrio/efeitos dos fármacos , Estrogênios não Esteroides/farmacologia , Feminino , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Camundongos , Morfogênese/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genéticaRESUMO
The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.
Assuntos
Fertilidade/fisiologia , Deleção de Genes , Útero/crescimento & desenvolvimento , Proteínas Wnt/metabolismo , Animais , Proliferação de Células , Implantação do Embrião/fisiologia , Feminino , Fertilidade/genética , Regulação da Expressão Gênica/fisiologia , Camundongos , Útero/citologia , Útero/metabolismo , Proteínas Wnt/genéticaRESUMO
It is estimated that infertility affects 15-20% of couples and can arise from female or male reproductive defects. Mouse models have ascribed roles to over 100 genes in the maintenance of female fertility. Although previous models have determined roles for apoptosis in male and female fertility, we find that compromised autophagy within the perinatal ovary, through the loss of Becn1 or Atg7, results in the premature loss of female germ cells. Becn1(+/-) ovaries have a 56% reduction of germ cells compared with control ovaries at post-natal day 1, whereas Atg7(-/-) ovaries lack discernable germ cells at this stage. Thus autophagy appears to be a cell survival mechanism to maintain the endowment of female germ cells prior to establishing primordial follicle pools in the ovary.
Assuntos
Autofagia , Fertilidade , Ovário/patologia , Óvulo/patologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Contagem de Células , Sobrevivência Celular , Feminino , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , RNA Mensageiro/metabolismoRESUMO
We evaluated the effect of different timing variations of an applied heat shock on parthenogenetically activated (PA) porcine embryos. PA embryos were heat shocked for 9 hr at 42 degrees C from either 0-9 hr post activation (hpa; 09HS), 13-22 hpa (1322HS), or 22-31 hpa (2231HS). Analysis of 24-hr cleavage rates (P < 0.0001), day 5 cell numbers (P < 0.005), day 7 blastocyst rates (P < 0.0001), and day 7 cell numbers (P < 0.05) showed that 09HS embryos developed more successfully in vitro than did all other treated and control embryos. In vitro fertilized (IVF) embryos were exposed to similar heat treatments as described for PA embryos, and embryos derived from somatic cell nuclear transfer (SCNT) were exposed only to the control and 09HS treatments to assess the effects of the different heat treatments on the timing of first cleavage and development to blastocyst. Embryos derived from both IVF and SCNT showed higher proportions of cleaved embryos on day 1 of development when heat shocked immediately after fertilization or fusion/activation as compared to NHS controls (P < 0.05). Blastocyst rates however, showed only modest (IVF; P = 0.089) or no (SCNT; P > 0.1) improvement as compared with control embryos. In summary, exposing PA embryos to elevated temperatures immediately after oocyte activation results in dramatically enhanced developmental potential. A thorough characterization of this phenomenon may yield findings that can serve to increase the efficiency with which PA, IVF, and SCNT embryos are produced in vitro.
Assuntos
Sobrevivência Celular , Oócitos/fisiologia , Zigoto/fisiologia , Animais , Feminino , Fertilização in vitro , Resposta ao Choque Térmico/fisiologia , Marcação In Situ das Extremidades Cortadas , Técnicas de Transferência Nuclear , Oócitos/citologia , Partenogênese , Gravidez , SuínosRESUMO
PURPOSE: BCL-X(L), an anti-apoptotic member of the BCL-2 family proteins and a cell death/survival checkpoint regulator, was shown to be upregulated in bright light-stressed mouse photoreceptors during an investigation of bright light-induced protein expression. To investigate the significance of BCL-X(L) upregulation in the bright light damage model, the Bcl-x gene was disrupted specifically in mouse rod photoreceptors, and the effect of Bcl-x disruption was characterized on retinal apoptosis, function, and morphology. METHODS: Rod-specific Bcl-x knockout mice, generated by mating mouse opsin promoter-controlled Cre mice with floxed Bcl-x mice, were subjected to bright light stress. Retinal apoptosis in the bright light-stressed conditional Bcl-x knockout mice was characterized with TUNEL, DNA fragmentation, and nuclear staining assays. Photoreceptor structural and functional integrity in the bright light-stressed conditional Bcl-x knockout mice was determined by measuring photoreceptor outer nuclear layer (ONL) thickness and electroretinography amplitudes. RESULTS: Disruption of Bcl-x in rod photoreceptors caused increased photoreceptor apoptosis, decreased retinal function, and decreased ONL thickness in bright light-stressed mice. CONCLUSIONS: The loss of BCL-X(L) increased rod photoreceptor susceptibility to bright light stress. Although the biochemical mechanism(s) of BCL-X(L) in photoreceptor death or survival has not been investigated extensively, results of the present study suggest that BCL-X(L), a cell survival/death checkpoint regulator, is involved in photoreceptor survival under bright light stress.
Assuntos
Apoptose , Luz , Lesões Experimentais por Radiação/etiologia , Degeneração Retiniana/etiologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Proteína bcl-X/fisiologia , Animais , Western Blotting , Fragmentação do DNA , Eletrorretinografia , Feminino , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Estresse Oxidativo , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Progesterone is a steroid hormone that is necessary to maintain pregnancy in mammals. We recently found that mice with a conditional deletion of Becn1/Beclin 1 specifically in the progesterone-synthesizing cells of the corpus luteum, had reduced progesterone synthesis and these mice failed to maintain pregnancy.(1) Furthermore, we identified that lipid storage and feedback through PRLR (prolactin receptor) and LHCGR (luteinizing hormone/choriogonadotropin receptor) were negatively affected by Becn1 deletion. BECN1 is necessary for the interaction of the 2 catalytic subunits of the class III phosphatidylinositol 3-kinase complex, PIK3C3, and PIK3R4, which are responsible for the generation of phosphatidylinositol 3-phosphate that is required for nucleation of the phagophore. Work from Sun et al. and Itakura et al. demonstrated that this BECN1 complex is also necessary for the fusion of autophagosomes and endosomes with lysosomes. Therefore, we suspected that ablating Becn1 in luteal cells would inhibit macroautophagy, hereafter referred to as autophagy. In support, we provide evidence that autophagic flux is reduced in our model. Thus, this study provides evidence that Becn1 is necessary for steroid production in murine luteal cells.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Corpo Lúteo/metabolismo , Trabalho de Parto Prematuro/metabolismo , Autofagia , Proteína Beclina-1 , Feminino , Humanos , Trabalho de Parto Prematuro/patologia , GravidezRESUMO
Recent attention on the early development of circadian rhythms has yielded several avenues of potential study regarding molecular and physiological rhythms in embryonic stem cells (ESCs) and their derivatives. While general guidelines of experimental design are-as always-applicable, there are certain idiosyncrasies with respect to experiments involving circadian rhythms that will be addressed. ESCs provide a number of challenges to the circadian biologist: growth rates are normally much higher than in established cell culture systems, the cells' innate drive towards differentiation and the lack of known synchronizing input pathways are a few examples. Some of these challenges can be addressed post hoc, such as normalization to total RNA or protein for transcript abundance studies. Most others, as outlined here, require special handling of the samples before and during experimentation in order to preserve any potential circadian oscillation that is present. Failure to do so may result in a disruption of endogenous oscillation(s) or, potentially worse, generation of an artificial oscillation that has no biological basis. This chapter begins with cultured ESCs, derived from primary blastocysts or in the form of cell lines, and outlines two methods of measuring circadian rhythms: the 2DG method of measuring glucose uptake (Sokoloff et al. J Neurochem 28:897-916, 1977) and real-time measurement of molecular rhythms using transgenic bioluminescence (Yoo et al. Proc Natl Acad Sci U S A 101:5339-5346, 2004).
Assuntos
Ritmo Circadiano , Células-Tronco Embrionárias/citologia , Medições Luminescentes/métodos , Animais , Relógios Biológicos , Técnicas de Cultura de Células , Linhagem Celular , Desoxiglucose/análise , Desoxiglucose/metabolismo , Células-Tronco Embrionárias/metabolismo , HumanosRESUMO
Tetraspanin CD151 interacts with laminin-binding integrins (i.e., α3ß1, α6ß1 and α6ß4) and other cell surface molecules to control diverse cellular and physiological processes, ranging from cell adhesion, migration and survival to tissue architecture and homeostasis. Here, we report a novel role of CD151 in maintaining the branching morphogenesis and activity of progenitor cells during the pubertal development of mammary glands. In contrast to the disruption of laminin-binding integrins, CD151 removal in mice enhanced the tertiary branching in mammary glands by 2.4-fold and the number of terminal end buds (TEBs) by 30%, while having minimal influence on either primary or secondary ductal branching. Consistent with these morphological changes are the skewed distribution of basal/myoepithelial cells and a 3.2-fold increase in proliferating Ki67-positive cells. These novel observations suggest that CD151 impacts the branching morphogenesis of mammary glands by upregulating the activities of bipotent progenitor cells. Indeed, our subsequent analyses indicate that upon CD151 removal the proportion of CD24(Hi)CD49f(Low) progenitor cells in the mammary gland increased by 34%, and their proliferating and differentiating activities were significantly upregulated. Importantly, fibronectin, a pro-branching extracellular matrix (ECM) protein deposited underlying mammary epithelial or progenitor cells, increased by >7.2-fold. Moreover, there was a concomitant increase in the expression and nuclear distribution of Slug, a transcription factor implicated in the maintenance of mammary progenitor cell activities. Taken together, our studies demonstrate that integrin-associated CD151 represses mammary branching morphogenesis by controlling progenitor cell activities, ECM integrity and transcription program.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Tetraspanina 24/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Glândulas Mamárias Animais/enzimologia , Camundongos , Morfogênese , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoAssuntos
Células Fotorreceptoras/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular/efeitos da radiação , Fragmentação do DNA , Eletrorretinografia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Luz/efeitos adversos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Estresse Oxidativo , Células Fotorreceptoras/patologia , Células Fotorreceptoras/fisiopatologia , Células Fotorreceptoras/efeitos da radiação , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/fisiopatologia , Recombinação Genética , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteína bcl-X/genéticaRESUMO
Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models.
Assuntos
Autofagia , Crescimento e Desenvolvimento , Animais , Apoptose/genética , Autofagia/genética , Humanos , Modelos Biológicos , Fagossomos/metabolismo , Estresse Fisiológico/genéticaRESUMO
The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The null hypothesis underlying the present study is that embryonic stem cells become rhythmic in both clock gene expression and glucose utilization only when allowed to spontaneously differentiate. Undifferentiated stem cells (ESCs, nâ=â6 cultures/timepoint for all experiments) were either maintained in their pluripotent state or released into differentiation (dESCs, nâ=â6 cultures/timepoint for all experiments). Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development.
Assuntos
Proteínas CLOCK/genética , Ritmo Circadiano/fisiologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Circadianas Period/genética , Animais , Relógios Biológicos/fisiologia , Proteínas CLOCK/metabolismo , Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Camundongos , Proteínas Circadianas Period/metabolismo , Periodicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Wnt genes are involved in critical developmental and growth processes. The present study comprehensively analyzed temporal and spatial alterations in Wnt and Fzd gene expression in the mouse uterus during peri-implantation of pregnancy. Expression of Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd2, Fzd4, and Fzd6 was detected in the uterus during implantation. Wnt4 mRNA was most abundant in the decidua, whereas Wnt5a mRNA was restricted to the mesometrial decidua during decidualization. Wnt7a, Wnt7b, and Wnt11 mRNAs were abundantly detected in the endometrial epithelia. The expression of Wnt7b was robust in the luminal epithelium (LE) at the implantation site on Gestational Day 5, whereas Wnt11 mRNA disappeared in the LE adjacent to the embryo in the antimesometrial implantation chamber but remained abundant in the LE. Wnt16 mRNA was localized to the stroma surrounding the LE on Day 4 and remained in the stroma adjacent to the LE but not in areas undergoing the decidual reaction. Fzd2 mRNA was detected in the decidua, Fzd4 mRNA was in the vessels and stroma surrounding the embryo, and Fzd6 mRNA was observed in the endometrial epithelia, stroma, and some blood vessels during implantation. Ovarian steroid hormone treatment was found to regulate Wnt genes and Fzd receptors in ovariectomized mice. Especially, single injections of progesterone stimulated Wnt11 mRNA, and estrogen stimulated Wnt4 and Wnt7b. The temporal and spatial alterations in Wnt genes likely play a critical role during implantation and decidualization in mice.
Assuntos
Implantação do Embrião/genética , Útero/metabolismo , Proteínas Wnt/genética , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Estradiol/farmacologia , Feminino , Receptores Frizzled/genética , Expressão Gênica/efeitos dos fármacos , Camundongos , Ovariectomia , Ovário/metabolismo , Gravidez , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Útero/efeitos dos fármacosRESUMO
Mitochondrial fission and fusion are linked to synaptic activity in healthy neurons and are implicated in the regulation of apoptotic cell death in many cell types. We developed fluorescence microscopy and computational strategies to directly measure mitochondrial fission and fusion frequencies and their effects on mitochondrial morphology in cultured neurons. We found that the rate of fission exceeds the rate of fusion in healthy neuronal processes, and, therefore, the fission/fusion ratio alone is insufficient to explain mitochondrial morphology at steady state. This imbalance between fission and fusion is compensated by growth of mitochondrial organelles. Bcl-x(L) increases the rates of both fusion and fission, but more important for explaining the longer organelle morphology induced by Bcl-x(L) is its ability to increase mitochondrial biomass. Deficits in these Bcl-x(L)-dependent mechanisms may be critical in neuronal dysfunction during the earliest phases of neurodegeneration, long before commitment to cell death.
Assuntos
Apoptose/fisiologia , Fusão de Membrana/fisiologia , Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Proteína bcl-X/genética , Animais , Metabolismo Energético/genética , Camundongos , Camundongos Knockout , Microscopia de Fluorescência/métodos , Mitocôndrias/patologia , Degeneração Neural/fisiopatologia , Neurônios/patologia , RatosRESUMO
Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.
Assuntos
Apoptose , Resposta ao Choque Térmico , Suínos/embriologia , Animais , Blastocisto , Feminino , Fertilização in vitro , Masculino , Oócitos/fisiologia , PartenogêneseRESUMO
Dendritic cells (DC) are important regulators of immune function, transporting Ags from the periphery to draining lymph nodes (dLN) where they prime Ag-specific T lymphocytes. The magnitude of the immune response generated depends upon the longevity of the Ag-bearing DC in lymphoid tissues. We hypothesized that the control of DC survival is regulated by the antiapoptotic factor bcl-x(L). Gene gun immunization of dual-expression DNA vaccines into a bcl-x(fl/fl) mouse resulted in the delivery of Ag, as well as selective deletion of the bcl-x gene in directly transfected, skin-residing DC. bcl-x-deficient DC failed to mount effective immune responses, and this corresponded to their rapid disappearance from the dLN due to apoptosis. We confirmed these results using RNA interference to specifically silence the antiapoptotic bcl-x(L) isoform in targeted skin-residing DC of C57BL/6 mice. In addition, delivery of bcl-x(L) in trans complemented the bcl-x deficiency in DC of bcl-x(fl/fl) mice, resulting in the maintenance of normal levels of Ag-bearing DC in the dLN. Taken together, our work demonstrates that the bcl-x(L) isoform is critical for survival of skin-derived, Ag-bearing DC in vivo.