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1.
Am J Obstet Gynecol ; 210(3): 252.e1-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24215857

RESUMO

OBJECTIVE: The purpose of this study was to test the hypothesis that the cytotoxic drug cis-platinum (CP) induces premature ovarian failure by reducing the viability of human granulosa cells. STUDY DESIGN: We incubated cultured human granulosa-luteal cells (GLCs) with varying concentrations of CP for 48 hours. Steroidogenesis and apoptosis were assessed by progesterone and estradiol, annexin V/propidium iodide, phase contrast, and transmission electron microscopy. RESULTS: CP caused impaired production of progesterone and estradiol in a dose- and a time-dependent fashion. The estradiol production was more pronounced than progesterone for each concentration of CP that was studied. The phase contrast microscopy of CP-treated GLCs showed loss of cell number with condensed nuclei. CP-induced apoptosis was maximum at 20 µg/mL compared with a 10-µg/mL concentration (79.9% ± 4.6% vs 58.3% ± 3.9%; P < .01). The hallmark of apoptosis (ie, nuclear condensation, cell size shrinkage) was seen in CP-treated cells by transmission electron microscopy. CONCLUSION: CP induces apoptosis of human GLCs in culture with impaired steroidogenesis, which may be one mechanism by which a CP-containing regime induces premature ovarian failure.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Estradiol/biossíntese , Células da Granulosa/efeitos dos fármacos , Células Lúteas/efeitos dos fármacos , Progesterona/biossíntese , Células Cultivadas , Feminino , Células da Granulosa/metabolismo , Humanos , Células Lúteas/metabolismo
2.
Biol Reprod ; 86(5): 153, 1-14, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22321830

RESUMO

In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.


Assuntos
Moléculas de Adesão Celular/fisiologia , Conexinas/fisiologia , Folículo Ovariano/fisiologia , Junções Aderentes/fisiologia , Junções Aderentes/ultraestrutura , Animais , Cálcio/fisiologia , Moléculas de Adesão Celular/ultraestrutura , Conexinas/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Feminino , Junções Comunicantes/fisiologia , Junções Comunicantes/ultraestrutura , Camundongos , Folículo Ovariano/ultraestrutura , Junções Íntimas/fisiologia , Junções Íntimas/ultraestrutura
3.
Magn Reson Med ; 68(2): 369-77, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22161549

RESUMO

This study aimed to characterize changes in lipid saturation using magnetic resonance spectroscopy of sensitive (HeLa) and resistant (C33A; Me180) cervical cancer cell lines following exposure to paclitaxel to explore lipid profiles as biomarkers of drug resistance. Spectra were acquired at 11.74 T. Flow cytometry, electron, and confocal microscopy assessed cellular morphology. Western blots assessed cytoplasmic phospholipase A(2) , fatty acid synthase, and acyl-CoA synthetase1 expression. After 24 h of paclitaxel exposure, >60% of cells showed mitotic arrest. At 48 h, HeLa cells showed apoptosis while C33A/Me180 cells showed normal morphology indicating resistance. MR-visible lipids increased significantly in all lines at 24 h with further increases at 48 h; resistant lines showed smaller increases than HeLa. Cytoplasmic phospholipase A(2) and fatty acid synthase levels were unchanged at 24 h and dropped at 48 h in HeLa; acyl-CoA synthetase1 was higher in Me180/C33A than in HeLa controls but did not increase significantly. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines; droplet size increased only in HeLa cells. Droplet number was >3-4× greater in apoptotic compared with mitotic-arrested cells. Apoptotic cells accumulate unsaturated fatty acids in large (relative to control) droplets; resistant lines accumulated smaller droplets with less triglycerides.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Lipídeos/análise , Espectroscopia de Ressonância Magnética/métodos , Mitose/efeitos dos fármacos , Paclitaxel/administração & dosagem , Biomarcadores Tumorais/análise , Feminino , Células HeLa , Humanos
4.
Biochem J ; 434(1): 49-60, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21118154

RESUMO

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.


Assuntos
Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Canalículos Biliares/fisiologia , Fígado/anatomia & histologia , Fígado/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Envelhecimento , Animais , Transporte Biológico/fisiologia , Membrana Celular , Colesterol/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética
5.
Cardiovasc Res ; 86(3): 443-51, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20083575

RESUMO

AIMS: Receptor-interacting protein 140 (RIP140) is a ligand-dependent cofactor for nuclear receptors that regulate networks of genes involved in cellular processes, including metabolism. An important role for RIP140 in metabolic control has been identified in RIP140 null mice, whose phenotypes include derepression of genes involved in energy mobilization or catabolism in adipocytes and a switch to more oxidative fibres in skeletal muscle. We hypothesized that ubiquitous expression of RIP140 would suppress metabolic processes, leading to defects in development or cellular function. METHODS AND RESULTS: The primary effect of exogenous expression of RIP140 mRNA (real-time PCR) and protein (western blotting) in transgenic mice is impaired postnatal heart function. There was rapid onset of cardiac hypertrophy and ventricular fibrosis, detected microscopically, in male RIP140 transgenic mice from 4 weeks of age, resulting in 25% mortality by 5 months. RIP140 exogenous expression in the heart leads to decreased mitochondria state III and state IV membrane potential and oxygen consumption. Quantitative PCR showed more than 50% reduced expression of genes involved in mitochondrial activity and fatty acid metabolism, including mitochondrial transcription factor A, cytochrome oxidase VIIa, cytochrome XII, CD36, medium-chain acyl dehydrogenase, and fatty acid transport protein, many of which are known targets for nuclear receptors, including peroxisome proliferator-activated receptors PPARalpha and PPARdelta and oestrogen-related receptors ERRalpha and ERRgamma. CONCLUSION: This study demonstrates that RIP140 is an important cofactor in postnatal cardiac function and that inhibition of the action of RIP140 may provide a model system to investigate specific interventions designed to prevent or delay the onset of cardiac disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cardiomegalia/metabolismo , Metabolismo Energético , Contração Miocárdica , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Envelhecimento , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Metabolismo Energético/genética , Feminino , Fibrose , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genótipo , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica/genética , Miocárdio/patologia , Proteínas Nucleares/genética , Proteína 1 de Interação com Receptor Nuclear , Consumo de Oxigênio , Fenótipo , RNA Mensageiro/metabolismo , Fatores Sexuais
6.
J Cell Sci ; 121(Pt 23): 3890-900, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19001500

RESUMO

The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.


Assuntos
Proliferação de Células , Células da Granulosa/citologia , Folículo Ovariano/crescimento & desenvolvimento , Animais , Forma Celular , Feminino , Células da Granulosa/ultraestrutura , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Mitose , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Ovário/metabolismo , Ovário/ultraestrutura , Células Tecais/metabolismo , Células Tecais/ultraestrutura
7.
Cell Metab ; 6(3): 236-45, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17767910

RESUMO

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Consumo de Oxigênio , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Células Cultivadas , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Mioblastos/citologia , Mioblastos/metabolismo , Miosinas/metabolismo , Proteínas Nucleares/genética , Proteína 1 de Interação com Receptor Nuclear , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , PPAR delta/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/metabolismo , Receptor ERRalfa Relacionado ao Estrogênio
8.
Biochem Biophys Res Commun ; 292(1): 102-8, 2002 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-11890678

RESUMO

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and "ciliogenic" cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.


Assuntos
Endométrio/química , Endométrio/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Microscopia Imunoeletrônica/métodos , Mucina-1/imunologia , Adulto , Endométrio/fisiologia , Epitélio/química , Epitélio/ultraestrutura , Epitopos/análise , Feminino , Humanos , Ciclo Menstrual , Mucina-1/análise
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