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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.


Assuntos
Actinas/metabolismo , Oócitos/metabolismo , Polimerização , Fuso Acromático/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Camundongos , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Oogênese/fisiologia
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.
FASEB J ; 34(8): 11292-11306, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32602619

RESUMO

Actin-interacting protein 1 (AIP1), also known as WD repeat-containing protein 1 (WDR1), is ubiquitous in eukaryotic organisms, and it plays critical roles in the dynamic reorganization of the actin cytoskeleton. However, the biological function and mechanism of AIP1 in mammalian oocyte maturation is still largely unclear. In this study, we demonstrated that AIP1 boosts ADF/Cofilin activity in mouse oocytes. AIP1 is primarily distributed around the spindle region during oocyte maturation, and its depletion impairs meiotic spindle migration and asymmetric division. The knockdown of AIP1 resulted in the gathering of a large number of actin-positive patches around the spindle region. This effect was reduced by human AIP1 (hAIP1) or Cofilin (S3A) expression. AIP1 knockdown also reduced the phosphorylation of Cofilin near the spindle, indicating that AIP1 interacts with ADF/Cofilin-decorated actin filaments and enhances filament disassembly. Moreover, the deletion of AIP1 disrupts Cofilin localization in metaphase I (MI) and induces cytokinesis defects in metaphase II (MII). Taken together, our results provide evidence that AIP1 promotes actin dynamics and cytokinesis via Cofilin in the gametes of female mice.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Citocinese/fisiologia , Metáfase/fisiologia , Oócitos/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Fosforilação/fisiologia , Fuso Acromático/metabolismo
5.
Anticancer Res ; 40(8): 4491-4504, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727779

RESUMO

BACKGROUND: Peroxiredoxin II (PRDX2) performs unique roles in cells. It can reduce peroxides through cysteine residues, and helps prevent the effects of oxidative stress on cells. It is closely related to the occurrence and development of various diseases, especially alcoholic liver injury and even liver cancer. The metabolism of alcohol in hepatocytes leads to the increase in the levels of reactive oxygen species (ROS), oxidative stress, injury, and apoptosis. Therefore, this study focused on the investigating the protection conferred by PRDX2 against alcohol-induced apoptosis of hepatocytes. MATERIALS AND METHODS: PRDX2 inhibition of alcohol-induced apoptosis in L02 hepatocytes was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescence microscopy, flow cytometry, western blotting and hematoxylin and eosin staining. RESULTS: The results showed that the levels of reactive oxygen species, protein kinase B, ß-catenin, B-cell lymphoma-2 (BCL2), BCL-XL, BCL2-associated X, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in PRDX2-silenced cells were increased significantly after the treatment of cells with ethanol. Similar results were obtained in an in vivo Prdx2-knockout mouse model of alcoholic liver injury. Therefore, PRDX2 may regulate the phosphorylation of the AKT signal protein by eliminating reactive oxygen species from cells, and it inhibits the downstream mitochondria-dependent apoptosis pathway, and, thereby, the apoptosis of cells. CONCLUSION: Thus, PRDX2 may be a potential molecular target for the prevention and treatment of alcoholic liver injury.


Assuntos
Etanol/efeitos adversos , Hepatócitos/citologia , Peroxirredoxinas/genética , Transdução de Sinais , Apoptose , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo
6.
Stem Cells ; 38(6): 727-740, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32083763

RESUMO

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson's disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD.


Assuntos
Células-Tronco Neurais/metabolismo , Neurotoxinas/metabolismo , Organoides/metabolismo , Doença de Parkinson/genética , Animais , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Doença de Parkinson/patologia
7.
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.

8.
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
9.
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
10.
Reprod Fertil Dev ; 31(3): 632, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31039976

RESUMO

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell-cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3'-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.

11.
Reproduction ; 157(3): 223-234, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817312

RESUMO

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.


Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Rad51 Recombinase/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Blastocisto/metabolismo , Reparo do DNA/efeitos dos fármacos , Feminino , Gravidez , Rad51 Recombinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Proteína Supressora de Tumor p53/metabolismo
12.
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.

13.
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
14.
Reprod Fertil Dev ; 31(2): 412-419, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30145997

RESUMO

Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell-cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3'-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Junções Íntimas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos , Ocludina/metabolismo , Poliadenilação , Proteínas de Ligação a RNA/genética , Suínos , Proteína da Zônula de Oclusão-1/metabolismo
15.
Sci Rep ; 8(1): 12581, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135500

RESUMO

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.


Assuntos
Autofagia , Meiose , Oócitos/citologia , Animais , Apoptose , Dano ao DNA , Feminino , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismo , Suínos
16.
J Cell Physiol ; 233(5): 4225-4234, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29058795

RESUMO

Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction.


Assuntos
Apoptose/genética , Estresse do Retículo Endoplasmático/genética , Ácido Graxo Sintases/genética , Proteína 1 de Ligação a X-Box/genética , Fator 4 Ativador da Transcrição/genética , Animais , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/genética , Feminino , Técnicas de Inativação de Genes , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Suínos , Fator de Transcrição CHOP/genética , Resposta a Proteínas não Dobradas/genética
17.
ChemMedChem ; 12(8): 580-589, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28296169

RESUMO

Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Oócitos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirróis/farmacologia , Animais , Compostos Azabicíclicos/farmacologia , Permeabilidade da Membrana Celular , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Oligopeptídeos/farmacologia , Organofosfatos/síntese química , Organofosfatos/farmacologia , Domínios Proteicos , Pirróis/síntese química , Pirróis/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Suínos , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/fisiologia
18.
PLoS One ; 11(12): e0168329, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27997591

RESUMO

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 µM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 µM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.


Assuntos
Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Glicólise/fisiologia , Meiose/fisiologia , Oócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Ácido Iodoacético/farmacologia , Meiose/efeitos dos fármacos , Oócitos/citologia , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Suínos
19.
PeerJ ; 4: e2454, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27672508

RESUMO

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

20.
PeerJ ; 4: e1914, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27077008

RESUMO

The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.

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