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1.
Environ Mol Mutagen ; 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31922297

RESUMO

As a member of parabens (PBs), Isobutylparaben (IBP) has a broad-spectrum antimicrobial activity and widely used in personal care products and cosmetics. Recent studies have indicated that usage of IBP poses a potential threat to reproductive health. In this study, we aimed to reveal the effects of acute exposure to IBP on the meiotic maturation of porcine cumulus oocyte complexes. Initial study showed that 200 µM of IBP significantly reduced the rate of the first polar body extrusion with no significant effect on cumulus cell expansion; however, 400 µM of IBP could significantly affect both. Further research revealed that abnormal spindles, misalignment chromosomes, and aberrant distributed actin filaments were detected in IBP-treated oocytes, which indicates that the cytoskeleton architecture of oocyte could be the target of IBP. At the same time, ROS level and apoptosis rate of oocyte were significantly increased by IBP exposure. Moreover, the levels of H3K9me3 and H3K27me3 were significantly induced in oocytes by IBP. Collectively, these results demonstrate that acute exposure to IBP could disrupt porcine oocyte maturation through affecting cytoskeleton, oxidative stress, viability and epigenetic modification. Environ. Mol. Mutagen. 2020. © 2020 Wiley Periodicals, Inc.

2.
J Cell Physiol ; 235(3): 2836-2846, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31535366

RESUMO

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

3.
Aging (Albany NY) ; 112019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31834867

RESUMO

Resveratrol (3,5,4'-trihydroxystilbene, RSV) is a natural potential anti-aging polyphenolic compound frequently used as a nutritional supplement against several diseases. However, the underlying mechanisms by which resveratrol regulates postovulatory aging of oocytes are still insufficiently known. In this study, we found that resveratrol could delay postovulatory aging and improve developmental competence of oocytes through activating selective mitophagy in the mouse. Resveratrol could maintain spindle morphology but it disturbed cortical granule (CG) distribution during oocyte aging. This might be due to upregulated mitophagy, since blocking mitophagy by cyclosporin A (CsA) treatment affected oocyte quality by damaging mitochondrial function and it decreased embryonic development. In addition, we also observed an involvement of FoxO3a in regulating mitophagy in aging oocytes following resveratrol treatment. Taken together, our results provide evidence that mitophagy induced by resveratrol is a potential mechanism to protect against postovulatory oocyte aging.

4.
Exp Cell Res ; : 111773, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31836472

RESUMO

In female meiosis, oocyte meiotic maturation is a form of asymmetric cell division, producing the first polar body and a large oocyte, in which the asymmetry of oocyte meiotic division depends on spindle migration and positioning, and cortical polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays an important role for asymmetric meiotic division in mouse oocyte. Our initial study demonstrated that WDR62 mainly co-localized with chromosomes during mouse oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not affect germinal vesicle breakdown (GVBD) but compromised the first polar body extrusion (PBE) with the large polar bodies generated, which is coupled with a higher incidence of spindle abnormality and chromosome misalignment. Further analysis concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap formation, which should be responsible for large polar body extrusion. Moreover WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but not expression level. In summary, our study defines WDR62 as an essential cytoskeletal regulator of spindle migration and asymmetric division during mouse oocyte meiotic maturation.

5.
Toxicol Sci ; 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31368505

RESUMO

Developments in chemotherapeutics have enhanced the survival rate of cancer patients, however adverse effects of chemotherapeutics on ovarian functions causes the fertility loss in young female cancer patients. Doxorubicin (DOX), as an anthracycline antitumor antibiotic, is extensively used to cure various malignancies. Recent studies have suggested that DOX can cause ovarian damage and affect the oocyte maturation, nevertheless the mechanism by which DOX on oocytes meiosis is poorly understood. In this study we explored the mechanism for DOX induced oocytes meiotic failure in vitro at human relevant exposure levels and time periods. Results described that DOX (100 nM) can interrupt the mouse oocytes meiotic maturation directly with reduced first polar body extrusion (PBE). Cell cycle analysis showed most oocytes were arrested at metaphase I (MI) stage. However, DOX treatment had no effect on spindle structure but chromosomal misalignment. We observed kinetochore-microtubule (K-MT) structure was affected and the spindle assemble checkpoint (SAC) was provoked after DOX treatment. Moreover, severe DNA damage was found in DOX treated oocytes indicated by the positive γ-H2A.X foci signal, which then may trigger oocytes early apoptosis. Besides, MII oocytes with disorganized spindle morphologies and misaligned chromosomes were observed after DOX treatment. In conclusion DOX have the potential to disrupt oocyte meiotic maturation through DNA damage induced meiotic arrest mediated by SAC activation. These findings can contribute to design the new therapies to alleviate DNA damage to preserve fertility for young female cancer patients with chemotherapeutics.

6.
Environ Mol Mutagen ; 60(9): 807-815, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31411769

RESUMO

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is a kind of additive flame retardants (FRs) and was found to affect early embryonic development in zebrafish; however, there are few studies to investigate whether TDCPP will disturb the development of early mouse embryos. In our studies, we used mouse embryos as models to study the toxicology of TDCPP on the early embryos. The results showed that TDCPP disturbed the development of early mouse embryos in a dose-dependent manner. 10 µM TDCPP decreased the blastocyst formation and 100 µM TDCPP was a lethal concentration for the mouse embryos. We proved that TDCPP was detrimental to embryonic development potential by increasing the reactive oxygen species level and inducing early apoptosis. Furthermore, TDCPP changed the DNA methylation patterns of imprinted genes in treated blastocysts. The methylation of H19 and Snrpn promoter regions was increased from 37.67% to 46.00% and 31.56% to 44.38% in treated groups, respectively. In contrast, Peg3 promoter region methylation was declined from 86.55% to 73.27% in treated embryos. Taken together, our results demonstrated that TDCPP could adversely impair the early embryonic development in mouse. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

7.
Chemosphere ; 237: 124410, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31362132

RESUMO

The profound influence of environmental chemicals on human health including inducing life-threatening gene mutation has been publicly recognized. Being a substitute for the extensively used endocrine-disrupting chemical BPA, Bisphenol AF (BPAF) has been known as teratogen with developmental toxicities and therefore potentially putting human into the risk of biological hazards. Herein, we deciphered the detrimental effects of BPAF on spermatogenesis and spermiotiliosis in sexual maturity of mice exposing to BPAF (5, 20, 50 mg/kg/d) for consecutive 28 days. BPAF exposure significantly compromises blood-testis barrier integrity and sperm quantity and quality in a dose-dependent manner. Sperms from BPAF exposure mice are featured by severe DNA damage, altered SUMOylation and ubiquitination dynamics and interfered epigenetic inheritance with hypermethylation of H3K27me3 presumably due to the aggregation of cellular reactive oxygen species (ROS). Furthermore, BPAF treatment (50 µM for 24 h) compromises cytoskeleton architecture and tight junction permeability in primary cultured Sertoli cells evidenced by dysfunction of actin regulatory proteins (e.g. Arp3 and Palladin) via activation of ERK signaling, thereby perturbing the privilege microenvironment created by Sertoli cells for spermatogenesis. Overall, our study determines BPAF is deleterious for male fertility, leading to a better appreciation of its toxicological features in our life.

8.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
9.
Methods Mol Biol ; 2045: 271-281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29511974

RESUMO

Stem cells have the potential to differentiate into specialized cell types under specific conditions in vivo or in vitro, which are used to cure many diseases related to aging. Somatic cell nuclear transfer (SCNT) can reprogram differential somatic cells into cloned embryos and embryonic stem cells can be derived from these cloned embryos. Recipient oocytes have healthier mitochondria and can improve the metabolism competence, lessen the ROS damage, and rejuvenate mitochondrial function of aged cells during reprogramming. Here, we describe a protocol to isolate aged somatic cells and reprogram them into embryonic stem cells by SCNT. These stem cells can be used to differentiate into regenerative somatic cells and replace the aged cells.

10.
Environ Mol Mutagen ; 60(3): 243-253, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30499614

RESUMO

Fluorene-9-bisphenol (BHPF), a substitute of bisphenol A (BPA) used in the production of the so-called "BPA-free" plastics, has now been shown to be released from commercial plastic bottles into drinking water and has strong anti-estrogenic activity in mice, which suggests that BHPF is also an environmental toxin. However, whether BHPF exposure has effects on mouse oocyte development is unknown. In this study, the influence of acute exposure to BHPF (50-150 µM, 12 hr) on mouse oocyte maturation and its possible mechanisms were investigated. Of note, 50-µM BHPF had no effects on the maturation of mouse oocytes, whereas 100- and 150-µM BHPF significantly blocked germinal vesicle breakdown and led to the failure of first polar body extrusion. Particularly, 100-µM BHPF exposure severely decreased the cellular adenosine triphosphate in a time-dependent manner, which finally brought out the loss of spindles. In addition, the actin cytoskeleton was also impaired. The defective mitochondrial dynamics and decreased mitochondrial DNA implied the damage of mitochondria in BHPF-treated oocytes. Increased PINK1, Beclin1, and LC3B protein level and decreased TOMM20 and TOMM17A protein level illustrated that mitophagy was induced, which also confirmed that BHPF exposure impaired the cellular mitochondria. Moreover, BHPF induced reactive oxygen species accumulation and early apoptosis. Oocyte quality was also impaired by BHPF exposure through altering histone modifications evidenced by increased H3K9me3 and H3K27me3 levels. Collectively, our results indicated that BHPF exposure disrupted mouse oocyte maturation and reduced oocyte quality through affecting cytoskeleton architecture, mitochondrial function, oxidative stress, apoptosis, and histone modifications. Environ. Mol. Mutagen. 60:243-253, 2019. © 2018 Wiley Periodicals, Inc.


Assuntos
Compostos Benzidrílicos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Fluorenos/toxicidade , Dinâmica Mitocondrial/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Fenóis/toxicidade , Citoesqueleto de Actina/patologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Beclina-1/metabolismo , DNA Mitocondrial/análise , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/patologia , Oócitos/citologia , Estresse Oxidativo/efeitos dos fármacos , Plásticos/análise , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo
11.
Cell Cycle ; 17(21-22): 2436-2446, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30382773

RESUMO

SURVIVIN is an essential chromosomal passenger complex (CPC) subunit and participates in cell division. In this study, we used porcine oocyte as a model to investigate the roles of Survivin during porcine oocyte maturation. Survivin was highly expressed in germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages oocytes, mainly localized in the GV at GV stage and on the chromosomes after GVBD. We have used RNA interference to specifically deplete Survivin in oocytes during in vitro maturation (IVM). Immunofluorescence assay showed that Survivin-depleted oocytes failed to produce polar body in meiosisⅠ (failed to complete cytokinesis), and they were arrested in metaphaseⅠwith misaligned chromosomes. The homologous chromosomes in Survivin-depleted oocytes could not be separated normally. Moreover, both the phosphorylation levels of Aurora B and the mRNA level of Mad2L1 related to spindle assembly checkpoint (SAC) was decreased in Survivin-depleted oocytes, which thus inhibited the degradation of Cyclin B1 (CCNB1) to complete meiosis. Taken together, we conclude that Survivin is an important mediator of centromere and midbody docking of Aurora-B as well as its activity and regulates SAC and MPF activity during meiosis in porcine oocytes.


Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos , Meiose , Oócitos/enzimologia , Survivina/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fosforilação , Transdução de Sinais , Fuso Acromático/enzimologia , Fuso Acromático/genética , Survivina/genética , Sus scrofa
12.
Exp Cell Res ; 371(2): 435-443, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30195030

RESUMO

Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment.


Assuntos
Cinetocoros/metabolismo , Meiose , Microtúbulos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cinetocoros/ultraestrutura , Camundongos , Microtúbulos/ultraestrutura , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oócitos/ultraestrutura , Fosforilação , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/ultraestrutura
13.
Nat Genet ; 50(5): 768, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29487399

RESUMO

In the version of this article originally published, the positions of Wenjie Shu and Qiang Wang in the author list were reversed and incorrect images were displayed in the HTML for Supplementary Figs. 1-12. The errors have been corrected in the HTML and PDF versions of the article.

14.
Biol Reprod ; 99(2): 308-318, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29546328

RESUMO

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Here we showed that autophagy was essential for the glycolysis switch and energy homeostasis in mouse granulosa cells under hypoxic condition. Our data indicated that hypoxia inducible factor-1α (HIF-1α) could be largely increased in developing follicles and this remarkable upregulation of HIF-1α triggered cell autophagy and glucose uptake. We found that blocking autophagy by Atg7 knockdown and 3-methyladenine (3-MA) treatment affected the glucose metabolism, with increased glycolytic enzyme activity and decreased ATP production. We also found enhanced lactate level, which was harmful to granulosa cells and could induce cell apoptosis. Thus, our findings highlight a protective role of HIF-1α-dependent autophagy for the granulosa cell glycolysis switch in both energy supply and cell survival.


Assuntos
Autofagia/fisiologia , Hipóxia Celular/fisiologia , Glicólise/fisiologia , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/farmacologia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Camundongos
15.
Nat Genet ; 50(3): 432-442, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29459681

RESUMO

Maternal obesity can impair embryo development and offspring health, yet the mechanisms responsible remain poorly understood. In a high-fat diet (HFD)-based female mouse model of obesity, we identified a marked reduction of Stella (also known as DPPA3 or PGC7) protein in oocytes. Starting with this clue, we found that the establishment of pronuclear epigenetic asymmetry in zygotes from obese mice was severely disrupted, inducing the accumulation of maternal 5-hydroxymethylcytosine modifications and DNA lesions. Furthermore, methylome-wide sequencing analysis detected global hypomethylation across the zygote genome in HFD-fed mice, with a specific enrichment in transposon elements and unique regions. Notably, overexpression of Stella in the oocytes of HFD-fed mice not only restored the epigenetic remodeling in zygotes but also partly ameliorated the maternal-obesity-associated developmental defects in early embryos and fetal growth. Thus, Stella insufficiency in oocytes may represent a critical mechanism that mediates the phenotypic effects of maternal obesity in embryos and offspring.


Assuntos
Anormalidades Congênitas/genética , Desenvolvimento Embrionário/genética , Obesidade/genética , Oócitos/metabolismo , Complicações na Gravidez , Proteínas Repressoras/genética , Animais , Anormalidades Congênitas/etiologia , Anormalidades Congênitas/metabolismo , Anormalidades Congênitas/patologia , Dieta Hiperlipídica/efeitos adversos , Embrião de Mamíferos , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/genética , Camundongos , Camundongos Obesos , Obesidade/complicações , Obesidade/patologia , Oócitos/patologia , Fenótipo , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/patologia , Proteínas Repressoras/metabolismo
16.
Biol Reprod ; 98(4): 449-464, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29325037

RESUMO

Understanding factors that regulate zygotic genome activation (ZGA) is critical for determining how cells are reprogrammed to become totipotent or pluripotent. There is limited information regarding how this process occurs physiologically in early mammalian embryos. Here, we identify a mediator complex subunit, MED13, as translated during mouse oocyte maturation and transcribed early from the zygotic genome. Knockdown and conditional knockout approaches demonstrate that MED13 is essential for ZGA in the mouse, in part by regulating expression of the embryo-specific chromatin remodeling complex, esBAF. The role of MED13 in ZGA is mediated in part by interactions with E2F transcription factors. In addition to MED13, its paralog, MED13L, is required for successful preimplantation embryo development. MED13L partially compensates for loss of MED13 function in preimplantation knockout embryos, but postimplantation development is not rescued by MED13L. Our data demonstrate an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during ZGA.


Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo Mediador/metabolismo , Oócitos/metabolismo , Animais , Cromatina/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Genoma , Complexo Mediador/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zigoto/metabolismo
17.
Environ Mol Mutagen ; 59(3): 223-233, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29285797

RESUMO

Sodium fluoride (NaF) is used as a medicine to prevent tooth decay; however, excessive NaF could cause a pathological damage to the health. Recent studies showed that NaF impaired mouse oocyte maturation, included of abnormal spindle configuration, actin cap formation, cortical granule-free domain formation, and the following development after fertilization. However, few studies used large animals as models to study the toxicology of NaF on oocytes maturation. We proposed a hypothesis that NaF would affect the nuclear and cytoplasmic maturation of porcine oocytes and DNA methylation pattern of imprinted genes in oocytes. Our results showed that NaF affected cumulus expansion, polar body emission, spindle morphology, cortical granule distribution, early apoptosis, and the following development after parthenogenetic activation during porcine oocyte maturation. Moreover, NaF increased the DNA methylation of NNAT and decreased its expression, which disturbed the glucose transport in oocytes. These results suggest that NaF impairs the porcine oocytes maturation epigenetically, which provides a new toxicological mechanism of NaF on the oocyte maturation. Environ. Mol. Mutagen. 59:223-233, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Proteínas do Tecido Nervoso/genética , Oócitos/metabolismo , Fluoreto de Sódio/farmacologia , Animais , Cariostáticos/farmacologia , Células Cultivadas , Feminino , Proteínas do Tecido Nervoso/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Suínos
18.
Int J Biol Sci ; 13(12): 1470-1478, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29230095

RESUMO

Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity. Additionally, evaluation of on- and off-target scoring algorithms was integrated into the CRISPR-offinder. The CRISPR-offinder has provided the bench biologist a rapid and efficient tool for identification of high quality target sites, and it is freely available at https://sourceforge.net/projects/crispr-offinder-v1-2/ or http://www.biootools.com.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Guia/genética , Software , Algoritmos , Desoxirribonuclease I/metabolismo , Edição de Genes , Células HEK293 , Humanos , Mutação
19.
Cell Cycle ; 16(22): 2220-2229, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28933982

RESUMO

Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study, we used porcine oocyte as a model to investigate the function of Chk1 during porcine oocyte maturation. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages, mainly localized in the cytoplasm at GV stage and moved to the spindle after germinal vesicle breakdown (GVBD). Chk1 depletion not only induced oocytes to be arrested at MI stage with abnormal chromosomes arrangement, but also inhibited the degradation of Cyclin B1 and decreased the expression of Mitotic Arrest Deficient 2-Like 1 (Mad2L1), one of spindle assembly checkpoint (SAC) proteins, and cadherin 1 (Cdh1), one of coactivation for anaphase-promoting complex/cyclosome (APC/C). Moreover, Chk1 overexpression delayed GVBD. These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at anaphase I (AI) and maintained the expression of Mad2L1 and Cdh1, which ensured that all chromosomes were accurately located in a line, and then oocytes passed metaphase I (MI) and AI and exited from the first meiotic division successfully. In addition, we proved that Chk1 had not function on GVBD of porcine oocytes, which suggested that maturation of porcine oocytes did not need the DNA damage checkpoint, which was different from the mouse oocyte maturation.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Oócitos/metabolismo , Animais , Quinase 1 do Ponto de Checagem/genética , Meiose/genética , Meiose/fisiologia , Metáfase/genética , Metáfase/fisiologia , Suínos
20.
Stem Cell Reports ; 9(2): 642-653, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28689997

RESUMO

The nucleosome, the fundamental structural unit of chromatin, is a critical regulator of gene expression. The mechanisms governing changes to nucleosome occupancy and positioning during somatic cell reprogramming remain poorly understood. We established a method for generating genome-wide nucleosome maps of porcine embryonic fibroblasts (PEF), reconstructed 1-cell embryos generated by somatic cell nuclear transfer (SCNT), and fertilized zygotes (FZ) using MNase sequencing with only 1,000 cells. We found that donor PEF chromatin, especially X chromosome, became more open after transfer into porcine oocytes and nucleosome occupancy decreased in promoters but increased in the genic regions. Nucleosome arrangements around transcriptional start sites of genes with different expression levels in somatic cells tended to become transcriptionally silent in SCNT; however, some pluripotency genes adopted transcriptionally active nucleosome arrangements. FZ and SCNT had similar characteristics, unlike PEF. This study reveals the dynamics and importance of nucleosome positioning and chromatin organization early after reprogramming.


Assuntos
Reprogramação Celular , Técnicas de Transferência Nuclear , Nucleossomos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Feminino , Fertilização In Vitro , Expressão Gênica , Masculino , Ligação Proteica , Análise de Sequência de DNA , Suínos , Sítio de Iniciação de Transcrição , Ativação Transcricional
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