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1.
Cell Cycle ; 20(2): 225-235, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33397186

RESUMO

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

2.
Front Cell Dev Biol ; 8: 602097, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33324650

RESUMO

Particulate matter (PM) is a general atmospheric pollutant released into the air by an anthropogenic and naturally derived mixture of substances. Current studies indicate that fine dust can result in different health defects, including endothelial dysfunction, asthma, lung cancer, cardiovascular diseases, uterine leiomyoma, deterioration in sperm quality, and overall birth impairment. However, the most prominent effects of PM10 (diameter < 10 µM) exposure on the female reproductive system, especially with respect to oocyte maturation, remain unclear. In the present study, maturing mouse oocytes were treated with PM10 and the phenotypes of the resulting toxic effects were investigated. Exposure to PM10 led to impairment of maturation capacity by inducing cell cycle arrest and blocking normal polar body extrusion during in vitro maturation and activation of fertilization of mouse oocytes. Additionally, defects in tubulin formation and DNA alignment were observed in PM10-treated oocytes during metaphase I to anaphase/telophase I transition. Moreover, PM10 induced reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and early apoptosis. Taken together, these results indicate that PM10 exposure leads to a decline in oocyte quality and affects the subsequent embryonic development potential of mammalian oocytes.

3.
In Vivo ; 34(4): 1823-1833, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606152

RESUMO

BACKGROUND/AIM: Picrasma quassioides (P. quassioides) is used in traditional Asian medicine widely for the treatment of anemopyretic cold, eczema, nausea, loss of appetite, diabetes mellitus, hypertension etc. In this study we aimed to understand the effect of P. quassioides ethanol extract on SiHa cervical cancer cell apoptosis. MATERIALS AND METHODS: The P. quassioides extract-induced apoptosis was analyzed using the MTT assay, fluorescence microscopy, flow cytometry and western blotting. RESULTS: P. quassioides extract induced cellular apoptosis by increasing the accumulation of cellular and mitochondrial reactive oxygen species (ROS) levels and inhibiting ATP synthesis. Pretreatment with N-Acetylcysteine (NAC), a classic antioxidant, decreased the intracellular ROS production and inhibited apoptosis. In addition, the P38 MAPK signaling pathway is a key in the apoptosis of SiHa cells induced by the P. quassioides extract. CONCLUSION: The P. quassioides extract exerts its anti-cancer properties on SiHa cells through ROS-mitochondria axis and P38 MAPK signaling. Our data provide a new insight for P. quassioides as a therapeutic strategy for cervical cancer treatment.


Assuntos
Picrasma , Neoplasias do Colo do Útero , Apoptose , Feminino , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Picrasma/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
4.
Asian-Australas J Anim Sci ; 33(10): 1579-1589, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32054159

RESUMO

OBJECTIVE: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. METHODS: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). RESULTS: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and ß-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. CONCLUSION: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

5.
In Vivo ; 33(4): 999-1010, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31280188

RESUMO

Infrastructure in animal husbandry refers to fundamental facilities and services necessary for better living conditions of animals and its economy to function through better productivity. Mainly, infrastructure can be divided into two categories: hard infrastructure and soft infrastructure. Physical infrastructure, such as buildings, roads, and water supplying systems, belongs to hard infrastructure. Soft infrastructure includes services which are required to maintain economic, health, cultural and social standards of animal husbandry. Therefore, the proper management of infrastructure in animal husbandry is necessary for animal welfare and its economy. Among various technologies to improve the quality of infrastructure, non-thermal plasma (NTP) technology is an effectively applicable technology in different stages of animal husbandry. NTP is mainly helpful in maintaining better health conditions of animals in several ways via decontamination from microorganisms present in air, water, food, instruments and surfaces of animal farming systems. Furthermore, NTP is used in the treatment of waste water, vaccine production, wound healing in animals, odor-free ventilation, and packaging of animal food or animal products. This review summarizes the recent studies of NTP which can be related to the infrastructure in animal husbandry.


Assuntos
Criação de Animais Domésticos , Gases em Plasma , Poluição do Ar , Ração Animal , Bem-Estar do Animal , Animais , Animais Domésticos , Ambiente Controlado , Água/análise , Água/química , Microbiologia da Água
6.
Sci Rep ; 9(1): 8774, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217533

RESUMO

Measurements of the three-dimensional (3D) structure of spermatozoon are crucial for the study of developmental biology and for the evaluation of in vitro fertilization. Here, we present 3D label-free imaging of individual spermatozoon and perform quantitative analysis of bovine, porcine, and mouse spermatozoa morphologies using refractive index tomography. Various morphological and biophysical properties were determined, including the internal structure, volume, surface area, concentration, and dry matter mass of individual spermatozoon. Furthermore, Holstein cows and Korean native cattle spermatozoa were systematically analyzed and revealed significant differences in spermatozoa head length, head width, midpiece length, and tail length between the two breeds. This label-free imaging approach provides a new technique for understanding the physiology of spermatozoa.


Assuntos
Imageamento Tridimensional , Espermatozoides/citologia , Animais , Bovinos , Masculino , Refratometria , Especificidade da Espécie , Espermatozoides/metabolismo
7.
Sci Rep ; 9(1): 8640, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201338

RESUMO

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) plays an important role in RNA processing via in m6A modification of pre-mRNA or pre-miRNA. However, the functional role of and relationship between m6A and hnRNPA2/B1 in early embryonic development are unclear. Here, we found that hnRNPA2/B1 is crucial for early embryonic development by virtue of regulating specific gene transcripts. HnRNPA2/B1 was localized to the nucleus and cytoplasm during subsequent embryonic development, starting at fertilization. Knockdown of hnRNPA2/B1 delayed embryonic development after the 4-cell stage and blocked further development. RNA-Seq analysis revealed changes in the global expression patterns of genes involved in transcription, translation, cell cycle, embryonic stem cell differentiation, and RNA methylation in hnRNPA2/B1 KD blastocysts. The levels of the inner cell mass markers OCT4 and SOX2 were decreased in hnRNPA2/B1 KD blastocysts, whereas that of the differentiation marker GATA4 was decreased. N6-Adenosine methyltransferase METTL3 knock-down caused embryonic developmental defects similar to those in hnRNPA2/B1 KD embryos. Moreover, METTL3 KD blastocysts showed increased mis-localization of hnRNPA2/B1 and decreased m6A RNA methylation. Taken together, our results suggest that hnRNPA2/B1 is essential for early embryogenesis through the regulation of transcription-related factors and determination of cell fate transition. Moreover, hnRNPA2/B1 is regulated by METTL3-dependent m6A RNA methylation.


Assuntos
Desenvolvimento Embrionário , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Mamíferos/embriologia , Mamíferos/metabolismo , Metiltransferases/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Metilação , Metiltransferases/genética , Camundongos Endogâmicos ICR , RNA/metabolismo , Interferência de RNA , Transcriptoma/genética
8.
Mol Reprod Dev ; 86(8): 972-983, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31136049

RESUMO

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.


Assuntos
Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Oócitos/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Oócitos/citologia , Suínos
9.
PeerJ ; 6: e5840, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30643672

RESUMO

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression.

10.
FASEB J ; 33(3): 4432-4447, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30557038

RESUMO

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis, and depletion of zinc impairs cell cycle control, asymmetric division, and cytokinesis in oocyte. We report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte impaired cortical and cytoplasmic actin formation. Spire is colocalized with zinc-containing vesicles via its zinc finger-containing Fab1, YOTB, Vac 1, EEA1 (FYVE) domain. Improper localization of Spire by zinc depletion or mutations in the FYVE domain impair cytoplasmic actin mesh formations and asymmetric division and cytokinesis of oocyte. All 3 major domains of the Spire are required for its proper localization and activity. After fertilization or parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire by controlling its localization in mammalian oocyte.-Jo, Y.-J., Lee, I.-W., Jung, S.-M., Kwon, J., Kim, N.-H., Namgoong, S. Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.


Assuntos
Citoesqueleto de Actina/fisiologia , Divisão Celular Assimétrica/fisiologia , Meiose/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Oócitos/metabolismo , Dedos de Zinco/fisiologia , Zinco/fisiologia , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Animais , Citocinese , Vesículas Citoplasmáticas/metabolismo , Feminino , Forminas/metabolismo , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Oócitos/citologia , Partenogênese/efeitos dos fármacos , Mutação Puntual , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Injeções de Esperma Intracitoplásmicas , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Estrôncio/farmacologia
11.
J Cell Sci ; 131(23)2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30404832

RESUMO

Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cellular events, and various actin-regulatory proteins modulate actin polymerization and depolymerization. Adenylyl cyclase-associated proteins (CAPs), highly conserved actin monomer-binding proteins, have been known to promote actin disassembly by enhancing the actin-severing activity of the ADF/cofilin protein family. In this study, we found that CAP1 regulated actin remodeling during mouse oocyte maturation. Efficient actin disassembly during oocyte maturation is essential for asymmetric division and cytokinesis. CAP1 knockdown impaired meiotic spindle migration and asymmetric division, and resulted in an accumulation of excessive actin filaments near the spindles. In contrast, CAP1 overexpression reduced actin mesh levels. CAP1 knockdown also rescued a decrease in cofilin family protein overexpression-mediated actin levels, and simultaneous expression of human CAP1 (hCAP1) and cofilin synergistically decreased cytoplasmic actin levels. Overexpression of hCAP1 decreased the amount of phosphorylated cofilin, indicating that CAP1 facilitated actin depolymerization via interaction with ADF/cofilin during mouse oocyte maturation. Taken together, our results provide evidence for the importance of dynamic actin recycling by CAP1 and cofilin in the asymmetric division of mouse female gametes.This article has an associated First Person interview with the first author of the paper.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Destrina/metabolismo , Oócitos/metabolismo , Serina Endopeptidases/metabolismo , Animais , Divisão Celular/fisiologia , Feminino , Camundongos , Oócitos/citologia
12.
FASEB J ; 32(2): 625-638, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970258

RESUMO

Mammalian oocytes lack a centriole that acts as a microtubule organization center (MTOC) in most somatic cells. During oocyte maturation, MTOCs undergo remodeling processes, including decondensation, fragmentation, and self-organization. However, the underlying mechanisms of MTOC remodeling in mouse oocytes are not well understood. We showed that two pericentriolar proteins, Cep192 and Cep152, play crucial roles during MTOC remodeling in mouse oocytes. Cep192 is present in MTOCs at all stages of oocyte maturation, and its depletion induces ablation of MTOCs, delay in spindle formation, and abnormal chromosomal alignment in spindles. In the case of Cep152, its localization on MTOCs is limited at the germinal vesicle stage and then disappears from the MTOCs after the germinal vesicle breakdown stage. Cep152 exclusion from MTOCs is involved in the fragmentation of MTOCs, and it is regulated by cyclin-dependent kinase 1 activity. Our results demonstrate the different roles of Cep192 and Cep152 in MTOC remodeling and a novel regulatory mechanism during meiotic spindle formation in mouse oocytes.-Lee, I.-W., Jo, Y.-J., Jung, S.-M., Wang, H.-Y., Kim, N.-H., Namgoong, S. Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Meiose/fisiologia , Centro Organizador dos Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas Cromossômicas não Histona/genética , Feminino , Camundongos , Oócitos/citologia , Fuso Acromático/genética
13.
PLoS Genet ; 13(6): e1006777, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28594822

RESUMO

Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.


Assuntos
Variação Genética , Genoma Helmíntico , Hibridização Genética , Poliploidia , Reprodução Assexuada , Tylenchoidea/genética , Animais , Elementos de DNA Transponíveis , Genoma Mitocondrial , Polimorfismo Genético , Seleção Genética
14.
Mol Hum Reprod ; 23(3): 166-176, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28364522

RESUMO

Study question: What is the function of Spindlin 1 (Spin1) in metaphase II stage oocytes in pigs? Summary answer: Depletion of Spin1 induces spontaneous oocyte activation and overexpression of Spin1 causes multinuclear formation through induction of DNA damage in porcine oocytes. What is known already: Little is known about the function of Spin1 in oocytes and embryos. In mouse oocytes, Spin1 is specifically expressed during gametogenesis and is essential for meiotic resumption. In somatic cells, Spin1 promotes cancer cell proliferation and activates WNT/T-cell factor signaling. Study design size, duration: After knockdown (KD) or overexpression of Spin1 in porcine MII-stage oocytes, MII maintenance was checked following additional culture for 24 h. Investigated parthenotes were cultured up to the four cell (72 h) or blastocyst (7 days) stages. Participants/materials, setting, methods: Spin1 was knocked down in porcine oocytes and embryos via microinjection of pig Spin1-targeting siRNA. For Spin1 overexpression, porcine Spin1-eGFP cRNA was generated. Additionally, for rescue experiments, cRNA encoding siRNA-resistant mouse Spin1 was added to the pig Spin1-targeting siRNA. For the overexpression and rescue experiments, microinjection and culture were performed using the same methods as the KD experiments. Main results and the role of chance: KD of Spin1 in MII-stage porcine oocytes reduced metaphase-promoting factor and mitogen-activated protein kinase activities, resulting in spontaneous pronuclear formation without calcium activation. However, the DNA damage response was triggered by Spin1 overexpression, generating the checkpoint protein γH2A.X. Furthermore, Spin1 overexpression blocked metaphase-anaphase transition and led to multinucleation in oocytes and embryos. Large scale data: None. Limitations, reasons for caution: This study is based on in vitro investigations with abnormal expression levels of Spin1. This may or may not accurately reflect the situation in vivo. Wider implications of the findings: Spin1 is essential to maintain MII arrest, but a high level of Spin1 induces DNA damage in oocytes and embryos. Thus, a system to accurately regulate Spin1 expression operates in porcine MII-stage oocytes and embryos. Study funding and competing interest(s): This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01057629). The authors declare no competing financial interests.


Assuntos
Blastocisto/metabolismo , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Metáfase , Proteínas Associadas aos Microtúbulos/genética , Oócitos/metabolismo , Fosfoproteínas/genética , Animais , Blastocisto/citologia , Cálcio/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica , Dano ao DNA , Embrião de Mamíferos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Suínos
15.
Oncotarget ; 8(24): 38631-38641, 2017 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-28418847

RESUMO

If no fertilization occurs at an appropriate time after ovulation, oocyte quality deteriorates rapidly as a process called postovulatory aging. Because the postovulatory aging of oocytes has detrimental effects on embryo development and offspring, many efforts have been made to prevent oocyte aging. Here we showed that quercetin prevented the decline in oocyte quality during postovulatory aging of oocytes. Quercetin treatment reduced aging-induced morphological changes and reactive oxygen species accumulation. Moreover, quercetin attenuated the aging-associated abnormalities in spindle organization and mitochondrial distribution, preventing decrease of SIRT expression and histone methylation. Quercetin also ameliorated the decrease in maturation-promoting factor activity and the onset of apoptosis during postovulatory aging. Furthermore, quercetin treatment during postovulatory aging improves early embryo development. Our results demonstrate that quercetin relieves deterioration in oocyte quality and improves subsequent embryo development.


Assuntos
Senescência Celular/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas Ligadas por GPI/metabolismo , Oócitos/fisiologia , Ovulação/efeitos dos fármacos , Quercetina/farmacologia , Sirtuína 1/metabolismo , Animais , Antioxidantes/farmacologia , Células Cultivadas , Feminino , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia , Oócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio , Sirtuína 1/genética , Espermatozoides/metabolismo
16.
Oncotarget ; 8(16): 26979-26991, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28439046

RESUMO

Protein phosphatase 2A regulatory subunit B55α (PP2A-B55α) has been studied in mitosis. However, its functions in mammalian meiosis and early embryonic development remain unknown. Here, we report that PP2A-B55α is critical for mouse oocyte meiosis and preimplantation embryo development. Knockdown of PP2A-B55α in oocytes led to abnormal asymmetric division, disordered spindle dynamics, defects in chromosome congression, an increase in aneuploidy, and induction of the DNA damage response. Moreover, knockdown of PP2A-B55α in fertilized mouse zygotes impaired development to the blastocyst stage. The impairment of embryonic development might have been due to induction of sustained DNA damage in embryos, which caused apoptosis and inhibited cell proliferation and outgrowth potential at the blastocyst stage. Overall, these results provide a novel insight into the role of PP2A-B55α as a novel meiotic and embryonic competence factor at the onset of life.


Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Oócitos/citologia , Oócitos/metabolismo , Proteína Fosfatase 2/metabolismo , Aneuploidia , Animais , Apoptose/genética , Blastocisto/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/genética , Transporte Proteico
17.
Biochim Biophys Acta ; 1863(12): 2993-3000, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27693251

RESUMO

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.


Assuntos
Segregação de Cromossomos/efeitos dos fármacos , Ciclina B/genética , Quinases Ciclina-Dependentes/genética , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Aneuploidia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Feminino , Regulação da Expressão Gênica , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Meiose/genética , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia , Oócitos/metabolismo , Fenilenodiaminas/farmacologia , Corpos Polares/metabolismo , Corpos Polares/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Pirimidinas/farmacologia , Transdução de Sinais , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura
18.
Mol Reprod Dev ; 83(9): 792-801, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27508507

RESUMO

Anillin is a scaffold protein that recruits several proteins involved in cleavage furrow formation during cytokinesis. The role of anilllin in symmetric cell divisions in somatic cells has been intensively studied, yet its involvement in cleavage furrow formation is still elusive. In this study, we investigated the role of anillin in mammalian oocyte maturation and cytokinesis. We found that anillin is localized around the nucleus during the oocyte germinal-vesicle stage, and spreads to the cytoplasm after germinal vesicle breakdown. Thereafter, anillin concentrates at the site of the cleavage furrow from anaphase I to metaphase II. Disruption of anillin activity by microinjecting oocytes with specific siRNAs resulted in a failure of polar body extrusion and asymmetric division, and caused abnormal chromosome segregation during anaphase I. Furthermore, pharmacological inhibition of myosin light chain using Y-27632 or ML-7 resulted in decreased anillin expression. Collectively, our data suggest that anillin is an essential intracellular component that maintains the integrity of asymmetric division in mouse oocytes. Mol. Reprod. Dev. 83: 792-801, 2016 © 2016 Wiley Periodicals, Inc.


Assuntos
Anáfase/fisiologia , Divisão Celular Assimétrica/fisiologia , Proteínas Contráteis/metabolismo , Metáfase/fisiologia , Oócitos/metabolismo , Animais , Proteínas Contráteis/genética , Feminino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia
19.
Sci Rep ; 6: 29204, 2016 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-27374327

RESUMO

The dynamic polymerization and depolymerization of actin filaments is essential for various cellular processes such as cell migration, rotation, cytokinesis, and mammalian oocyte maturation. Tropomodulin 3 (Tmod3) binds to the slow-growing (pointed) ends of the actin filament, thereby protecting the filament from depolymerization. However, the roles of Tmod3 in mammalian oocyte maturation remain elusive. Tmod3 mRNA and protein is present at all stages of mouse oocyte maturation. Tmod3 protein is mainly localized in the cytoplasm and appears enriched near the chromosome during maturation. By knocking down or ectopically overexpressing Tmod3, we confirmed that Tmod3 regulate the level of the intracytoplasmic actin mesh and asymmetric spindle migration. Expression of N-terminal Tmod3 (correspond to 1-155 amino acids), which contains the tropomyosin-binding site, results in decreased density of the actin mesh, thereby demonstrating the importance of the interaction between tropomyosin and tropomodulin for the maintenance of the actin mesh. Taken together, these findings indicate that Tmod3 plays crucial roles in oocyte maturation, presumably by protecting the actin filament from depolymerization and thereby controlling the density of the cytoplasmic actin mesh.


Assuntos
Divisão Celular Assimétrica , Diferenciação Celular , Oócitos/citologia , Oócitos/metabolismo , Tropomodulina/metabolismo , Actinas/metabolismo , Animais , Citoplasma/metabolismo , Proteínas Fetais/metabolismo , Forminas , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Transporte Proteico , Fuso Acromático/metabolismo , Tropomiosina/metabolismo
20.
Sci Rep ; 6: 20408, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26842404

RESUMO

The SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) proteins constitute the linker of nucleoskeleton and cytoskeleton (LINC) complex on the nuclear envelope. To date, the SUN1/KASH5 complex is known to function as meiotic-specific factors. In this study, gene-silencing methods were used to explore the roles of SUN1 and KASH5 in mouse oocytes after prophase. SUN1 was detected throughout the nucleus; however, KASH5 was dispersed through the cell. After germinal vesicle breakdown (GVBD), SUN1 and KASH5 migrated during spindle formation and localized to the spindle poles at the MII stage. Most oocytes were arrested at the germinal vesicle (GV) stage after depletion of either SUN1 or KASH5. The DNA damage response was triggered in SUN1-depleted oocytes and thus gave rise to the G2/M checkpoint protein, p-CHK1. Oocytes that underwent GVBD had relatively small and abnormal spindles and lower levels of cytoplasm F-actin mesh. Immunofluorescence results also indicated the dislocation of pericentrin and P150(Glued) after SUN1 or KASH5 depletion. Furthermore, KASH5 localized exclusively near the oocyte cortex after SUN1 depletion, but SUN1 localization was unaffected in KASH5-depleted oocytes. Taken together, the results suggest that SUN1 and KASH5 are essential factors in the regulation of meiotic resumption and spindle formation.


Assuntos
Proteínas de Ciclo Celular/genética , Citoesqueleto/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Oócitos/citologia , Prófase , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Dano ao DNA , Feminino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/fisiologia , Fuso Acromático/genética , Fuso Acromático/metabolismo
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