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1.
Int J Mol Sci ; 23(14)2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35887161

RESUMO

O-linked ß-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy-D-gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism.


Assuntos
Acetilglucosamina , N-Acetilglucosaminiltransferases , Acetilglucosamina/metabolismo , Animais , Bovinos , Feminino , Glucose/metabolismo , Células da Granulosa/metabolismo , Homeostase , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteína X Associada a bcl-2/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
2.
Theriogenology ; 188: 28-36, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35661480

RESUMO

The expression and function of bone morphogenetic protein 4 (BMP4) gene in bovine cumulus cells (CCs) was investigated to reveal the mechanisms by which it regulated cell apoptosis and proliferation. The mRNA and protein expression of BMP4 were detected using quantitative PCR (qPCR) and immunofluorescence staining in CCs. The effective siRNAs against BMP4 gene were screened using qPCR and western blotting. The mRNA expression levels of apoptosis-related genes and proliferation-related genes were estimated by qPCR after knocking-down the BMP4 gene in bovine CCs. Cell apoptosis, proliferation and cell cycle were measured with Annexin V-FITC, CCK-8 and propidium iodide staining by flow cytometry. Results showed that the BMP4 gene was expressed and its protein was in the cytoplasm and nuclei of bovine CCs. The BMP4 knockdown increased the cell apoptosis rate and upregulated the mRNA levels of apoptosis genes CASPASE-3 and BAX with downregulation of the anti-apoptosis gene BCL-2 (P < 0.05). The proliferation rate declined and the mRNA expression levels of proliferation-related genes PCNA, CDC42 and CCND2 were downregulated in the bovine CCs with BMP4 low expression (P < 0.05). The BMP4 knockdown significantly increased the percentage of G0/G1 phase cells while decreased that of S phase cells. Therefore, the expression of BMP4 and its biological functions on the cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. BMP4 knockdown induced cell apoptosis, cell cycle arrest and inhibited proliferation of bovine CCs.


Assuntos
Apoptose , Células do Cúmulo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/farmacologia , Bovinos , Proliferação de Células , Células do Cúmulo/metabolismo , Feminino , RNA Mensageiro/metabolismo
3.
Theriogenology ; 161: 65-73, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33296745

RESUMO

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P < 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3, BAX and BAX/BCL-2 ratio (P < 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12, two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P < 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed.


Assuntos
Apoptose , Células do Cúmulo , Animais , Bovinos , Proliferação de Células , Células do Cúmulo/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Metilação
4.
Reprod Domest Anim ; 55(9): 1103-1114, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32542809

RESUMO

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.


Assuntos
Ácido Ascórbico/farmacologia , Fertilização in vitro/veterinária , Licopeno/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Fertilização in vitro/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Pré-Seleção do Sexo/veterinária
5.
Reprod Fertil Dev ; 32(7): 676-689, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32317092

RESUMO

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.


Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Criopreservação/veterinária , Metilação de DNA/fisiologia , Técnicas de Maturação in Vitro de Oócitos , Sequenciamento Completo do Genoma/veterinária , Animais , Metilação de DNA/genética , Feminino , Fertilização in vitro/veterinária , Análise de Célula Única/métodos , Análise de Célula Única/veterinária , Sequenciamento Completo do Genoma/métodos
6.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409031

RESUMO

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.


Assuntos
Bovinos/fisiologia , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Capacitação Espermática , Animais , Apoptose , Feminino , Fertilização in vitro/veterinária , Masculino , Melatonina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo
7.
J Pineal Res ; 66(1): e12532, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30320949

RESUMO

Paraquat (PQ), a broad-spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ-induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro-produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic-related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ-exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ-induced p38 mitogen-activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl-histone H3 lysine 4 (H3K4me3) and trimethyl-histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ-induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.


Assuntos
Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Paraquat/toxicidade , Animais , Antioxidantes/metabolismo , Bovinos , Feminino , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Sci Rep ; 8(1): 7603, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29765076

RESUMO

The aim of this study was to test the effects of five different concentrations (0, 10-3, 10-4, 10-5, and 10-6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10-3 M and 10-4 M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10-3 M and 10-4 M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10-4 M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.


Assuntos
Fertilização in vitro/veterinária , Peroxidação de Lipídeos/efeitos dos fármacos , Resveratrol/farmacologia , Espermatozoides/citologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Proteína X Associada a bcl-2/genética , Proteína bcl-X/genética
9.
J Pineal Res ; 64(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28833478

RESUMO

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10-7 , 10-9 , and 10-11  mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization-related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.


Assuntos
Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Animais , Bovinos , Células Cultivadas , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
Sci Rep ; 7(1): 10652, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28878377

RESUMO

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 µM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.


Assuntos
Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Íons/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Rutênio Vermelho/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Ácido Egtázico/farmacologia , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , RNA Mensageiro/genética
11.
Oncotarget ; 8(3): 4656-4667, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-27999197

RESUMO

Lipopolysaccharide disturbs the secretion of gonadotropin, endometrial function and implantation efficiency. However, there is little information regarding the effects of lipopolysaccharide on cyclic ovary activity, especially oocyte maturation. Therefore, we aimed to investigate the effects of lipopolysaccharide on the maturation potential of bovine oocytes. We found that lipopolysaccharide exposure significantly decreased the first polar body extrusion rate and delayed the cell cycle progression. The abnormal spindle rate was significantly increased in lipopolysaccharide treatment group, accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase (p-MAPK). Moreover, lipopolysaccharide treatment significantly increased intracellular reactive oxygen species (ROS) levels and the early apoptotic rate in oocytes. The pro-apoptotic caspase-3 and Bax mRNA levels and caspase-3 protein level were significantly increased, whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were significantly decreased in lipopolysaccharide exposure group. Furthermore, the dimethyl-histone H3 lysine 4 (H3K4me2) level was significantly increased, while the DNA methylation (5-mC) and dimethyl-histone H3 lysine 9 (H3K9me2) levels were markedly decreased in oocytes treated with lipopolysaccharide. In conclusion, lipopolysaccharide exposure inhibits the maturation potential of bovine oocytes by affecting cell cycle, cytoskeletal dynamics, oxidative stress, and epigenetic modifications.


Assuntos
Epigênese Genética/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Oócitos/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Animais , Bovinos , Ciclo Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
12.
Mol Reprod Dev ; 83(11): 993-1002, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27653174

RESUMO

We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10-5 and 10-3 M melatonin led to higher plasma membrane integrity, mitochondrial activity, and acrosome integrity, and significantly decreased intracellular ROS levels (P < 0.05). The blastocyst development rate of in vitro-produced bovine embryos originating from 10-3 M melatonin-treated spermatozoa was significantly higher, while the incidence of apoptotic nuclei in blastocysts was markedly lower than for embryos from any other group (P < 0.05). CASP3 and BAX mRNA abundance were significantly reduced whereas BCL2, XIAP, and CAT transcript abundance were significantly increased in embryos produced from spermatozoa treated with 10-3 M melatonin; GPX4 expression, however, was comparable in all treatment groups. Thus, 10-3 M melatonin can improve the quality of bovine frozen-thawed semen. These beneficial effects appear to influence preimplantation embryos, given the correlation with its anti-apoptotic and anti-oxidative properties. Mol. Reprod. Dev. 83: 993-1002, 2016 © 2016 Wiley Periodicals, Inc.


Assuntos
Acrossomo/metabolismo , Técnicas de Cultura Embrionária , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro , Melatonina/farmacologia , Animais , Bovinos , Feminino , Masculino , Preservação do Sêmen
13.
J Pineal Res ; 60(2): 132-41, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26485053

RESUMO

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.


Assuntos
Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Melatonina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Oócitos/metabolismo , Animais , Caspase 3/metabolismo , Bovinos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Oócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
14.
Theriogenology ; 84(5): 716-23, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26055309

RESUMO

To determine the beneficial effects and mechanisms of exogenous glutathione (GSH) during IVC on the embryonic development, bovine IVF zygotes were cultured in medium containing different concentrations of GSH, and the rate of cleavage and blastocyst development, total cell number of blastocysts, the inner cell mass:total cell number ratio, and intracellular GSH and reactive oxygen species concentrations were investigated. Gene expressions associated with embryonic development and GSH metabolism were measured using quantitative real-time polymerase chain reaction. At the concentrations of 1, 3, and 5 mM, GSH significantly increased the blastocyst rate and embryo quality. The highest blastocyst rate (51%) and the best embryo quality appeared in the 3-mM GSH treatment. Intracellular content of GSH of embryos at the two- to four-cell stage significantly increased, and the reactive oxygen species level decreased accordingly with 3-mM GSH treatment, but no significant differences were found in the four- to eight-cell stage and blastocysts. Gene expression analysis of the embryo regulator genes (OCT4, SOX2, and KLF4), GSH synthesis genes (GCLM, GCLC, and GSS), and GSH utilization genes (GSTP, GSTM, and GPX) showed that the GSH had no significant impact on these genes. In conclusion, exogenous GSH during IVC improved developmental potential and quality of bovine IVF embryos, which was probably caused by the ability of GSH to maintain the redox balance.


Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/veterinária , Glutationa/farmacologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização in vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Glutationa/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
15.
Zygote ; 23(4): 525-36, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24869483

RESUMO

This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10-9 M melatonin (DOs + MT), cumulus-oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.


Assuntos
Blastocisto/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Blastocisto/fisiologia , Proteína Morfogenética Óssea 15/genética , Bovinos , Células Cultivadas , Células do Cúmulo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Folículo Ovariano/citologia , Partenogênese , Espécies Reativas de Oxigênio/metabolismo
16.
Zygote ; 22(3): 348-55, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23369698

RESUMO

We investigated the effect mouse cumulus cells (mCCs) on the in vitro maturation (IVM) and developmental potential of bovine denuded germinal vesicle oocytes (DOs). Cumulus-oocyte complexes (COCs), DOs and DOs cocultured with either mCCs (DOs + mCCs) or bovine cumulus cells (bCCs; DOs + bCCs) were subjected to IVM. The meiosis II (MII) rates of DOs, glutathione (GSH) contents, zona pellucida (ZP) hardening and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression levels of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) in MII oocytes were measured using quantitative real-time polymerase chain reaction (PCR). mCCs significantly increased the MII rate of DOs from 53.5 ± 3.58% to 69.67 ± 4.72% (p < 0.05) but had no effect on the GSH content (2.17 ± 0.31 pmol/oocyte with mCCs, 2.14 ± 0.53 pmol/oocyte without mCCs). For the DOs + mCCs group, the BMP-15 and GDF-9 expression levels were significantly higher and the ZP dissolution time was significantly lower (162.49 ± 12.51 s) than that of the DOs group (213.95 ± 18.87 s; p < 0.05). The blastocyst rate of the DOs + mCCs group (32.56 ± 4.94%) was similar to that of the DOs group (31.75 ± 3.65%) but was significantly lower than that of the COCs group (43.52 ± 5.37%; p < 0.05). In conclusion, mCCs increased the MII rate of DOs and expression of certain genes in MII oocytes, and decreased the ZP hardening of MII oocytes, but could not improve their GSH content or developmental potential.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Animais , Blastocisto/fisiologia , Proteína Morfogenética Óssea 15/genética , Bovinos , Técnicas de Cocultura , Células do Cúmulo , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Fator 9 de Diferenciação de Crescimento/genética , Meiose , Camundongos , Oócitos/citologia , Partenogênese , Zona Pelúcida/metabolismo
17.
Yi Chuan ; 35(5): 587-94, 2013 May.
Artigo em Chinês | MEDLINE | ID: mdl-23732664

RESUMO

After meiosis, round spermatid develops into mature sperm through metamorphosis. During this stage, most cytoplasm in the germ cell is gradually lost. The histones associated with chromatin are replaced by transition proteins and eventually transformed into protamines. Thus, the spermatid chromatin is stringently packaged and highly concentrated. It was thought that the transcription activity of spermatid is lost and RNAs are absent in spermatid. Nevertheless, many types of transcripts are detected in recent years, including the transcripts needed during chromatin repackaged and some small RNAs, etc. Because histones in the nuclear are not replaced entirely, and there are some active sites on the chromatin, we conjectured that spermatid has some transcription activity, and this activity is regulated by hormone and epigenetic modification. These RNAs may be the residues in the spermatogenesis, or timely expressed during spermiogenesis. A deep study on gene transcription in spermiogenesis will help understand the genetic characteristics and provide the theoretic basis for reproductive control using male gamete. This article reviewed recent advances in spermiogenesis at gene transcription level and proposed the future research directions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Espermatogênese , Espermatozoides/citologia , Transcrição Gênica , Animais , Humanos , Masculino , Espermátides/citologia , Espermátides/metabolismo , Espermatozoides/metabolismo
18.
Fertil Steril ; 100(1): 256-61, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23548937

RESUMO

OBJECTIVE: To investigate the effect of vitrification on Dnmt1o, Hat1, and Hdac1 promoter CpG island methylation patterns and messenger RNA (mRNA) expression levels in mouse metaphase II (MII) oocytes. DESIGN: In vitro study. SETTING: Academic institution. ANIMAL(S): Kunming white mice. INTERVENTION(S): After vitrification, surviving mouse MII oocytes subjected to methylation and expression analysis with fresh oocytes used as a control. MAIN OUTCOME MEASURE(S): Expression levels of mRNA as measured by real-time reverse-transcriptase polymerase chain reaction of methylation patterns of the CpG islands in the Dnmt1o, Hat1, and Hdac1 promoters analyzed by bisulfite mutagenesis and sequencing. RESULT(S): The methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, and Hdac1 were not statistically significantly when comparing vitrified oocytes and fresh oocytes. The expression levels of Hat1 and Hdac1 mRNA were not statistically significantly different in comparing in vitrified oocytes and fresh oocytes. The expression of Dnmt1o mRNA was statistically significantly lower in vitrified oocytes compared with fresh oocytes. CONCLUSION(S): Vitrification did not statistically significantly alter the methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, or Hdac1, but did statistically significantly decrease the expression of Dnmt1o mRNA in mouse MII oocytes.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/biossíntese , Histona Acetiltransferases/biossíntese , Histona Desacetilase 1/biossíntese , Oócitos/enzimologia , Vitrificação , Animais , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Regulação para Baixo/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Histona Acetiltransferases/antagonistas & inibidores , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Metáfase/genética , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese
19.
Am J Chin Med ; 40(3): 481-94, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22745065

RESUMO

Hepatic stellate cells (HSCs) undergo activation during the development of liver fibrosis. Transcription factor myocyte enhancer factor (MEF2) 2C plays a key role in this process. In the present study, we investigated the effect of hydroxysafflor yellow A (HSYA) on hepatic fibrosis and further investigated potential mechanisms in vivo. Sprague-Dawley rats were administered with CCl(4) together with or without HYSA for 12 weeks. The effect of HYSA on hepatic fibrosis was evaluated using hematoxylin-eosin and Van Gieson staining. Messenger RNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or immunohistochemistry. Our results revealed that CCl(4) treatment induced micronodular hepatic fibrosis with a pronounced deposition of collagen fibers. Treatment with HYSA resulted in a significant decrease in fibrosis, protein expression of α-SMA, and MEF-2C gene expression. This was accompanied by a decreased expression of Tß-RI, Tß-RII, MEKK3, MEK5, and phosphorylation of ERk5. HYSA alone had no effect on the measured parameters. Our findings demonstrate that HSYA protected, at least in part, the rat liver from CCl(4)-caused fibrogenesis through inhibition of hepatic stellate cell (HSC) activation, attenuation of transforming growth factor beta (TGF-ß) signaling. HSYA may become a novel and promising agent for the inhibition of hepatic fibrosis.


Assuntos
Carthamus/química , Chalcona/análogos & derivados , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Cirrose Hepática Experimental/tratamento farmacológico , Fígado/efeitos dos fármacos , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Fitoterapia , Quinonas/uso terapêutico , Actinas/metabolismo , Animais , Tetracloreto de Carbono , Chalcona/farmacologia , Chalcona/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colágeno/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/citologia , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Fatores de Transcrição MEF2 , Masculino , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Fosforilação , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Quinonas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
20.
Fertil Steril ; 98(1): 222-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22624671

RESUMO

OBJECTIVE: To determine the effect of vitrification and 5-aza-2'-deoxycytidine (5-aza-dC) on the methylation levels of the putative imprinted control region (ICR) of H19 and H19 expression in bovine two-cell embryos and their derived blastocysts. DESIGN: Experimental animal study. SETTING: Academic institution. ANIMAL(S): Abattoir-derived bovine ovaries. INTERVENTION(S): Vitrified two-cell embryos were cultured in vitro to blastocysts with 0.01 µM 5-aza-dC (5-aza-dC group) or without 5-aza-dC (vitrification group). Fresh embryos and their derived blastocysts were used as control. MAIN OUTCOME MEASURE(S): Putative ICR methylation of H19 was measured by bisulfate mutagenesis and sequencing, blastocyst development rate; total cell number were determined; and H19 expression was measured by real-time reverse transcriptase-polymerase chain reaction (PCR). RESULT(S): Vitrification significantly increased putative ICR methylation of H19 in two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 in vitrified two-cell embryos and their derived blastocysts. The H19 expression level was significantly higher in blastocysts from the 5-aza-dC group than the vitrification group. The blastocyst development rate and total cell number in the 5-aza-dC and vitrification groups were similar. CONCLUSION(S): Putative ICR methylation levels of H19 significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 and increased H19 expression in blastocysts derived from vitrified two-cell embryos.


Assuntos
Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , RNA não Traduzido/genética , Animais , Azacitidina/farmacologia , Bovinos/embriologia , Células Cultivadas , Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/metabolismo , Decitabina , Avaliação Pré-Clínica de Medicamentos , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Impressão Genômica/efeitos dos fármacos , Região de Controle de Locus Gênico/efeitos dos fármacos , Região de Controle de Locus Gênico/genética , RNA Longo não Codificante , Vitrificação
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