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Theriogenology ; 140: 44-51, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31437668


Lysosome, an important organelle in eukaryotes, can sequester macromolecules submitted by the endocytosis and autophagy pathways for degradation and recycling. Massive macromolecular turnover is also vital to the growth and development of mammalian oocytes. However, the functional role of lysosomes in the meiotic maturation of mammalian oocytes remains largely unexplored. Here, by treating in vitro matured porcine cumulus-oocyte complexes (COCs) with chloroquine (CQ), a lysosome inhibitor, we showed that regardless of CQ concentration, lysosomal inhibition affected neither the extrusion of the first polar body (PB1), nor the ROS levels. However, CQ treatment dramatically decreased the rates of oocytes with normal chromosome alignment and cytoskeleton organization (P < 0.05), but boosted the rates of oocytes with apoptosis (P < 0.05). Subsequently, after pathenogenetic activation or in vitro fertilization, the death or fragmentation rates of oocytes treated by CQ (both 35 µM and 45 µM) were significantly higher (P < 0.05), whereas the rates of embryo cleavage, embryos developed to blastocysts, and average blastomere number per blastocyst, were all significantly lower (P < 0.05), respectively. Furthermore, CQ (35 µM) treatment activated the autophagy pathway by elevating the LC3 II/I ratio. Taken together, lysosomes could affect porcine oocyte maturation and subsequent developmental capacity partially through the chromosome organization/cytoskeleton assembly and autophagy/apoptosis pathways.

Theriogenology ; 121: 160-167, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30165304


Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.

Metilação de DNA , Oócitos/citologia , Animais , Betaína/farmacologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Cicloleucina/farmacologia , Meiose/genética , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Suínos/embriologia
J Anim Sci ; 96(8): 3358-3369, 2018 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-29800308


Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90's relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90's effect on pig oocyte meiotic maturation. Endogenous Hsp90α was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90α protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90α by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90α can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes.

Proteínas de Choque Térmico/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Suínos/fisiologia , Animais , Feminino , Oócitos/fisiologia
Sci Rep ; 8(1): 6132, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29666467


L-ascorbic acid (Vitamin C) can enhance the meiotic maturation and developmental competence of porcine oocytes, but the underlying molecular mechanism remains obscure. Here we show the role of ascorbic acid in regulating epigenetic status of both nucleic acids and chromatin to promote oocyte maturation and development in pigs. Supplementation of 250 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) during in vitro maturation significantly enhanced the nuclear maturation (as indicated by higher rate of first polar body extrusion and increased Bmp15 mRNA level), reduced level of reactive oxygen species, and promoted developmental potency (higher cleavage and blastocyst rates of parthenotes, and decreased Bax and Caspase3 mRNA levels in blastocysts) of pig oocytes. AA2P treatment caused methylation erasure in mature oocytes on nucleic acids (5-methylcytosine (5 mC) and N 6 -methyladenosine (m6A)) and histones (Histone H3 trimethylations at lysines 27, H3K27me3), but establishment of histone H3 trimethylations at lysines 4 (H3K4me3) and 36 (H3K36me3). During the global methylation reprogramming process, levels of TET2 (mRNA and protein) and Dnmt3b (mRNA) were significantly elevated, but simultaneously DNMT3A (mRNA and protein), and also Hif-1α, Hif-2α, Tet3, Mettl14, Kdm5b and Eed (mRNA) were significantly inhibited. Our findings support that ascorbic acid can reprogram the methylation status of not only DNA and histone, but also RNA, to improve pig oocyte maturation and developmental competence.

Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Epigênese Genética/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Suínos/crescimento & desenvolvimento , Animais , Proteína Morfogenética Óssea 15/genética , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Feminino , Oócitos/citologia , Oócitos/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Suínos/genética , Suínos/metabolismo
J Biol Chem ; 293(5): 1767-1780, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29222335


The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.

Proteínas de Ciclo Celular , Sequenciamento de Nucleotídeos em Larga Escala , Meiose/fisiologia , Oócitos/metabolismo , Transcriptoma/fisiologia , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Feminino , Oócitos/citologia , Suínos
Huan Jing Ke Xue ; 35(2): 651-6, 2014 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-24812960


The advanced treatment of petrochemical secondary wastewater by ozone- aerated biological filter was carried out in this study. The effect of pH on ozonation and the removal of COD and UV254 by the ozone-aerated biological filter combined process were investigated. In addition, the variation of relative molecular mass distribution of organics and the characteristics of three-dimensional fluorescence spectra of the wastewater were also investigated. The results showed that the suitable operating conditions of the ozonation unit were: ozone dosage 10 mg x L(-1), contact time 4 min and slightly alkaline pH. Ozonation can transfer macromolecular organics into small molecular organics, resulting in a 15% increase in the percentage of the organics with small relative molecular mass (less than 1 000). The biodegradability of the petrochemical secondary effluent was significantly improved by ozonation, making it more suitable for the treatment by aerated biological filter. The removal efficiency of COD and UV254 were 40.8% and 45.8% when the hydraulic retention time was 3 hours and the gas to water ratio was 3:1 for BAF. The average COD of the petrochemical wastewater was 86.5 mg x L(-1) while the average COD of the effluent of the combined process was 49.4 mg x L(-1) when it was operated under optimal conditions.

Filtração/métodos , Ozônio/química , Eliminação de Resíduos Líquidos/métodos , Biodegradação Ambiental , Indústrias Extrativas e de Processamento , Águas Residuárias/química