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1.
J Biol Chem ; 288(38): 27619-27637, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23921385

RESUMO

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Complexos Multiproteicos/metabolismo , Plectina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Núcleo Celular/genética , Citoesqueleto/genética , Células HeLa , Humanos , Complexos Multiproteicos/genética , Mutação , Plectina/genética , Estrutura Terciária de Proteína
2.
Reprod Sci ; 17(2): 102-15, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20130288

RESUMO

After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.


Assuntos
Caspase 6/metabolismo , Cromatina/metabolismo , Lamina Tipo A/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Animais , Apoptose/fisiologia , Feminino , Camundongos , Oócitos/citologia , Oogênese/fisiologia
3.
Reprod Toxicol ; 29(1): 93-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19874886

RESUMO

For the first time, the effects of low doses of gamma-radiation were studied on folliculogenesis and on isolated oocytes. After irradiation of adult mice, even at the lowest dose, a drastic loss of primordial follicles was observed in serial sections of ovaries, with, in opposite, no effect on the other follicle stages. Moreover, oocytes freshly recovered from the largest antral follicles of irradiated adult ovaries exhibited significantly less regular Ca(2+) oscillations than controls. Finally, in vitro folliculogenesis demonstrated a smaller diameter of preantral follicles recovered from irradiated juvenile ovaries compared to control, and an increase in follicle atresia. Further on, PLC-beta1 localization was not affected in the enclosed oocytes whereas chromatin configuration revealed that a quarter of them had prematurely resumed meiosis or was fragmented. These results raise the question of the risk of genetic and teratogenic effects on women submitted to chronic exposure even of very low radiation.


Assuntos
Raios gama/efeitos adversos , Oócitos/efeitos da radiação , Folículo Ovariano/fisiologia , Folículo Ovariano/efeitos da radiação , Animais , Cálcio/análise , Radioisótopos de Cobalto , Relação Dose-Resposta à Radiação , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/química , Oócitos/crescimento & desenvolvimento , Irradiação Corporal Total
4.
Dev Dyn ; 237(12): 3892-903, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19035350

RESUMO

Several studies have shown that apoptotic pathways control fragmentation of unfertilized ovulated oocyte, induced by doxorubicin. But very few have investigated the basis of this process, from prophase I to later stages. Our results revealed the presence of caspase-2(L), caspase-9, and caspase-3 in their zymogen and cleaved forms in the oocyte before meiosis resumption. Caspase-2(L) and caspase-9 were detected in the nucleus of GV-oocytes in a distribution related to chromatin configuration. The inhibition of caspase activity by Z-VAD-fmk accelerated the transition from metaphase I to metaphase II, and caspase-9 and caspase-3 were detected along the meiotic spindle. Surprisingly, Western blot analysis revealed that the three cleaved caspases were present in similar amounts in healthy and fragmented oocytes and caspase inhibition did not prevent doxorubicin-induced apoptosis. Our results suggest that, if cleaved, caspases may be dispensable for final oocyte death and they could be involved in regulating the maturation process.


Assuntos
Caspase 2/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Diferenciação Celular , Oócitos/citologia , Oócitos/enzimologia , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Caspase 2/genética , Caspase 3/genética , Caspase 9/genética , Inibidores de Caspase , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cinética , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Transcrição Gênica/genética
5.
Reprod Sci ; 15(7): 661-72, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18836131

RESUMO

The dynamics of the subcellular distribution of PLCbeta1 was investigated during meiosis competence acquisition and meiosis resumption in relation to oocyte diameter and to nonsurrounded-nucleolus or surrounded-nucleolus chromatin configurations. Oocytes collected after both in vivo and in vitro folliculogenesis were studied. In both conditions, at the beginning of the process, most oocytes exhibited a nuclear PLCbeta 1 associated with a nonsurrounded-nucleolus chromatin configuration. Then at the final stage of the process, the factors shifted mainly toward a cytoplasmic PLCbeta1 and a surrounded-nucleolus chromatin configuration, typical of a preovulatory fertilizable oocyte. Additionally, only germinal vesicle oocytes with a diameter > 75 microm, and exhibiting cytoplasmic PLC beta1 distribution and surrounded-nucleolus chromatin configuration, resumed meiosis. Our findings demonstrate a strong correlation between oocyte diameter, chromatin configuration, and PLCbeta1 localization. Thus, PLCbeta1 localization appears to be a key factor determining the progressive acquisition of meiotic competence and final resumption of meiosis.


Assuntos
Meiose/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Fosfolipase C beta/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/enzimologia , Folículo Ovariano/citologia , Folículo Ovariano/enzimologia , Gravidez
6.
Toxicology ; 247(2-3): 80-7, 2008 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-18407394

RESUMO

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.


Assuntos
Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Urânio/toxicidade , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/patologia , Oócitos/fisiologia , Folículo Ovariano/patologia , Folículo Ovariano/fisiologia , Urânio/farmacocinética
7.
Crit Rev Eukaryot Gene Expr ; 17(4): 259-69, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17725492

RESUMO

As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.


Assuntos
Isoenzimas/metabolismo , Oócitos/enzimologia , Fosfatidilinositóis/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Cálcio/metabolismo , Feminino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipase C beta
8.
Cell Signal ; 15(11): 1003-10, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14499343

RESUMO

We aimed to determine whether the breakdown of the germinal vesicle of the mouse oocyte and the nuclear import of phospholipase C-beta1 were calcium-dependent. We chelated Ca2+ ions with BAPTA-dextran at different times after the release of the oocyte from the ovarian follicle, i.e. after meiosis resumption has started, and we studied the effects on the kinetics of germinal vesicle breakdown, and on the migration of phospholipase C-beta1. We discriminate between two key-periods of calcium-sensitivity during the process of meiosis resumption. During the first hour, changes in the cytosolic Ca2+ especially promoted the migration of phospholipase C-beta1 into the nucleus, whereas changes in the nuclear concentration of Ca2+ were not implicated. Moreover, at this time, the cytosolic calcium pathway is PLC-beta1-dependent. By contrast, during the second hour following the onset of meiosis resumption, and thus just previous GVBD, the PLC-beta1-dependent Ca2+ signals in both cellular compartments were equally necessary for the resumption of meiosis. This particular period of the meiotic process corresponds to the moment when the phospholipase C-beta1 has strongly migrated into the nucleus. Our results highlight also the role played by the nucleus during the second key-period in the control of the GVBD via a Ca2+-dependent pathway.


Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Ácido Egtázico/análogos & derivados , Isoenzimas/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Citoplasma/metabolismo , Ácido Egtázico/metabolismo , Feminino , Camundongos , Microscopia Confocal , Membrana Nuclear/metabolismo , Oócitos/citologia , Fosfolipase C beta , Transporte Proteico , Vesículas Secretórias/metabolismo
9.
Zygote ; 10(1): 59-64, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11964092

RESUMO

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.


Assuntos
Sinalização do Cálcio , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Envelhecimento/fisiologia , Animais , Técnicas de Cultura , Feminino , Gonadotropinas/metabolismo , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Folículo Ovariano/citologia
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