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/metabolismoRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.
Assuntos
Apoptose/efeitos dos fármacos , Blastocisto/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melatonina/farmacologia , Paraquat/efeitos adversos , Praguicidas/efeitos adversos , Animais , Blastocisto/patologia , Caspase 3/metabolismo , Bovinos , Feminino , Paraquat/farmacologia , Praguicidas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
The epidermal growth factor receptor (EGFR), a transmembrane protein receptor, is a member of ErbB family with signal-transducing tyrosine kinase activity. After combined with the ligand, EGFR homologous or heterologous dimers are formed to induce intracellular signal transduction, activate downstream signal transduction pathways, and then produce a series of biological effects. RAF/MEK/RAS/ERK pathway is relevant to cell proliferation, differentiation and apoptosis; while PDK1/AKT /PI3K pathway is involved in cell migration and adhesion. EGFR can promote the maturity of pulmonary type II epithelial cells and the synthesis and secretion of pulmonary surfactant. EGFR shows the effect on mammal lungs in a time-space and dose-dependent manner. The down-regulated expression of it will lead to immature lung development, while the over-expression can promote the cell proliferation, invasion and metastasis of the lung cancer cells. This paper reviewed advances in the study for EGFR and its signal pathway, as well as the relationship among EGFR, atelectasis and lung cancer.
Assuntos
Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/crescimento & desenvolvimento , Transdução de Sinais , Animais , Receptores ErbB/genética , Regulação da Expressão Gênica , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologiaRESUMO
This study was designed to examine the recovery of mitochondrial function and endogenous antioxidant systems in vitrified oocytes during extended incubations. After 16 hr of in vitro maturation, bovine meiosis-II oocytes were vitrified, and then surviving oocytes were cultured an additional 8 hr. ATP content, ATP synthase activity, expression of ATP synthase F0 subunit 6 (ATP6) and 8 (ATP8) genes, and reactive oxygen species (ROS) levels were investigated in the vitrified oocytes during this additional period (4 or 8 hr). The results showed that: (1) the ATP content and ATP synthase activities in vitrified oocytes at 8 hr post-warming (754.6 fmol, 25.9 nmol NADH/min/mg) were significantly higher than in oocytes immediately warmed (568.3 fmol, 8.7 nmol NADH/min/mg), but still lower than in control oocytes (901.5 fmol, 30.7 nmol NADH/min/mg); (2) the relative expression of ATP6 and ATP8 was initially down-regulated in oocytes when they were first warmed, increased by 4 hr post-warming, and were again down-regulated by 8 hr post-warming; (3) ROS levels in oocytes at 0, 4, and 8 hr post-warming were significantly higher than in control oocytes; and (4) after parthenogenetic activation, the blastocyst rate of oocytes at 8 hr post-warming (26.7%) was significantly higher than that of oocytes immediately warmed (16.9%). These results indicated that mitochondrial function and endogenous antioxidant systems recovered significantly better in vitrified-thawed bovine oocytes with 8 hr of additional incubation, but they did not achieve the activity levels found in fresh oocytes.
Assuntos
Antioxidantes/fisiologia , Técnicas de Cultura de Células/métodos , Mitocôndrias/fisiologia , Oócitos/fisiologia , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Antioxidantes/metabolismo , Blastocisto/metabolismo , Blastocisto/fisiologia , Bovinos , Feminino , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , VitrificaçãoRESUMO
Vitrification had a significantly negative impact on the mitochondrial function of bovine oocytes. However, 40 µg/mL cyclosporine pretreatment before vitrification contributed greatly to maintaining mitochondrial membrane potential and adenosine triphosphate content, decreasing reactive oxygen species level, and thereby increasing the developmental ability of vitrified oocytes.